CN110536719A - Cyclic dinucleotide derivatives for the treatment of cancer - Google Patents

Abstract

Provided herein are compounds useful for treating cancer.

Description

Cyclic dinucleotide derivatives for the treatment of cancer

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the following patent applications: US Provisional Patent Application No. 62/460,562, filed February 17, 2017; US Provisional Patent Application No. 62/479,169, filed March 30, 2017; August 29, 2017 US Provisional Patent Application No. 62/551,645, filed August 29, 2017; US Provisional Patent Application No. 62/551,647, filed August 29, 2017; and US Provisional Patent Application No. 62/551,668, filed August 29, 2017. All of these applications are incorporated herein by reference as if fully rewritten.

Background technique

STING (stimulator of interferon genes) is a signaling molecule in the cytosol in the innate response to dsDNA. Deletion of STING has been reported in various human cancers. Furthermore, in melanoma (Xia T et al, "Recurrent Loss of STINGSignaling in Melanoma Correlates with Susceptibility to Viral Oncolysis" Cancer Res. 2016) and colon cancer (Xia T et al, "Deregulation of STING Signaling in Colorectal Carcinoma Constrains DNA Damage Responses" Dysregulation of STING signaling in human cancers has also been reported in and Correlates With Tumorigenesis" Cell Rep. 2016;14:282-97). Interestingly, in these studies, the results of genomic analysis showed that the loss of expression of STING was not due to gene deletion or mutation, but through epigenetic changes (Xia, Cancer Res. 2016; Xia, Cell Rep. 2016). Evidence obtained from studies in mouse models also supports the cancer-protective activity of STING. STING knockout mice display defects in tumor control (Woo SR et al., "STING-dependent cytosolic DNA sensing mediates innate immunerecognition of immunogenic tumors" Immunity 2014;41:830-42).

In addition, the role of STING in protecting ontogeny has been demonstrated in several mouse spontaneous models, including glioma (Ohkuri T et al., "Protective role of STING against gliomagenesis: Rationaluse of STING agonist in anti-glioma immunotherapy" Oncoimmunology. 2015;4:e999523) and colon cancer (Zhu Q et al., "Cutting edge: STING mediates protection against colorectal tumorigenesis by governing the magnitude of intestinalinflammation" J. Immunol. 2014;193:4779-82). This antitumor effect may be due to its ability to counteract the overactivation of NF-kB and STAT3 (Okihuri 2015). Activation of the STING pathway has also shown potent activity in preclinical mouse tumor models (Woo 2014; Chandra D et al., "STING ligand c-di-GMP improves cancervaccination against metastatic breast cancer" Cancer Immunol Res. 2014;2: 901-10; Corrales L et al, "Direct Activation of STING in the Tumor MicroenvironmentLeads to Potent and Systemic Tumor Regression and Immunity" Cell Rep. 2015;11:1018-30; Curran E et al, "STING Pathway Activation Stimulates Potent Immunityagainst Acute Myeloid Leukemia "Cell Rep. 2016; 15:2357-66; Tang CH et al., "Agonist-Mediated Activation of STING Induces Apoptosis in Malignant B Cells" Cancer Res. 2016;76:2137-52). This antitumor activity may be due to disruption of the tumor vasculature, which then induces an adaptive immune response (Corrales L et al., "The host STING pathway at the interface of cancer and immunity" J. Clin. Invest. 2016;126:2404 -11) cause. Therefore, direct stimulation of STING in the tumor microenvironment by agonists may represent a new approach to the treatment of multiple cancer types.

SUMMARY OF THE INVENTION

Embodiments may provide compounds of Formula I:

₍ wherein P1 is phosphorus as depicted above in the lower left and P2 is phosphorus in the upper right as depicted above), the _compound has the substituents and stereochemistry shown in 71 below, or a pharmaceutically acceptable one thereof Salt. Indicates a single or double bond.

When the phosphorus atom has four different substituents, the phosphorus atom is the stereocenter. SpSp/RpRp/SpRp/RpSp refer to the indicated phosphorous stereochemistry.

Table 1

Embodiments may further provide compounds of Formula II:

The compounds have substituents and stereochemistry as shown in Table 2 below, or a pharmaceutically acceptable salt thereof. When the phosphorus atom has four different substituents, the phosphorus atom is the stereocenter.

Table 2

Compound number R_4a geometry Stereochemical configuration (P₁,P₂) 13 -OH trans S,R 14 -OH trans R,R 15 -F trans S,R 16 -F trans R,R

Embodiments may provide compounds of formula (III):

or a pharmaceutically acceptable salt thereof, wherein

R _1a is selected from the group consisting of -H, halogen, -C _1-6 alkyl, -N ₃ , -CH ₂ R _2a , -NHR _2a or -OR _2a ;

R _2a is selected from the group consisting of -H, -C _1-6 alkyl, or R _2a and R ₃ taken together represent -CH ₂ -, -CH ₂ CH ₂ -, -CH(CH ₂ Y ₁ )- or -CH ₂ CH(CH ₂ Y ₁ )-;

Y ₁ is selected from the group consisting of: -H; -OH optionally substituted with -C _1-3 alkyl; -NH ₂ optionally substituted with C _1-3 alkyl; optionally substituted with Allyl substituted with: (i)-OH, (ii) _-OCH3 , (iii) Ci- _3alkyl , or (iv) halogen; vinyl optionally substituted with: (i)-OH, (ii) _-OCH3 , (iii) _{Ci_3alkyl} , or (iv) halogen; or _-CH3 optionally substituted with: (i)-OH, ( ii) -OCH ₃ , (iii) C _1-3 alkyl, or (iv) halogen;

R _1b is selected from the group consisting of -H, halogen, -C _1-6 alkyl, -OR _2b , -NHR _2b , or with R ₁₁ to form C _4-6 cycloalkyl or C _4-6 hetero cycloalkyl;

R _2b is selected from the group consisting of -H or -C _1-6 alkyl;

R ₃ is -H; -F; -C _1-6 alkyl optionally substituted with: (i) C _1-3 alkyl, (ii) -OH, (iii) halogen, or (iv) )-OCH ₃ ; -C _2-6 alkenyl optionally substituted with: (i) C _1-3 alkyl, (ii)-OH, (iii) halogen, or (iv)-OCH ₃ ; C _2-6 alkynyl optionally substituted with: (i) C _1-3 alkyl, (ii)-OH, (iii) halogen, or (iv)-OCH ₃ ; or optionally -OC _1-6 alkyl substituted with: (i) C _1-3 alkyl, (ii) -OH, (iii) halogen, or (iv) -OCH ₃ ; or R ₃ is attached to R _2a ;

R _4a is selected from the group consisting of -H, halogen, -C _1-6 alkyl, -N ₃ , -CH ₂ R _5a , -NHR _5a or -OR _5a ;

R _5a is selected from the group consisting of -H, -C _1-6 alkyl, or R _5a and R ₆ taken together represent -CH ₂ -, -CH ₂ CH ₂ -, -CH(CH ₂ Y ₂ )- or -CH ₂ CH(CH ₂ Y ₂ )-;

Y ₂ is selected from the group consisting of: -H; -OH optionally substituted with -C _1-3 alkyl; -NH ₂ optionally substituted with C _1-3 alkyl; Allyl substituted with: (i)-OH, (ii) _-OCH3 , (iii) Ci- _3alkyl , or (iv) halogen; vinyl optionally substituted with: (i)-OH, (ii) _-OCH3 , (iii) _{Ci_3alkyl} , or (iv) halogen; or _-CH3 optionally substituted with: (i)-OH, ( ii) -OCH ₃ , (iii) C _1-3 alkyl, or (iv) halogen;

R _4b is selected from the group consisting of -H, halogen, -C _1-6 alkyl, -OR _5b , -NHR _5b , or with R ₁₂ to form C _4-6 cycloalkyl or C _4-6 hetero cycloalkyl;

R _5b is selected from the group consisting of -H or -C _1-6 alkyl;

R ₆ is -H; -F; -C _1-6 alkyl optionally substituted with: (i) C _1-3 alkyl, (ii) -OH, (iii) halogen, or (iv) )-OCH ₃ ; -C _2-6 alkenyl optionally substituted with: (i) C _1-3 alkyl, (ii)-OH, (iii) halogen, or (iv)-OCH ₃ ; C _2-6 alkynyl optionally substituted with: (i) C _1-3 alkyl, (ii)-OH, (iii) halogen, or (iv)-OCH ₃ ; or optionally -OC _1-6 alkyl substituted with: (i) C _1-3 alkyl, (ii) -OH, (iii) halogen, or (iv) -OCH ₃ ; or R ₆ is attached to R _5a ;

R ₁₁ is -H or -C _1-6 alkyl, or R ₁₁ and R _1b form a ring;

R ₁₂ is -H or -C _1-6 alkyl, or R ₁₂ and R _4b form a ring;

R ₂₅ and R ₁₈ are the same or different, and are independently selected from the group consisting of -H, C _1-3 alkyl, which is saturated or unsaturated, and optionally by C _1-3 alkyl , -OH, halogen or -OCH ₃ substitution;

Z ₁ and Z ₂ are the same or different, and are independently selected from -O-, -NH-, -N(C _1-3 alkyl), -CH ₂ - and -S-;

P ₁ and P ₂ each independently have an S or R stereochemical configuration;

X _1a and X _2a are the same or different, and are independently selected from O or S;

X _1b and X _2b are the same or different, and are independently selected from the group consisting of -OR ₁₉ , -NR ₁₉ R ₂₀ and -SR ₁₉ -,

wherein R ₁₉ and R ₂₀ are each independently selected from the group consisting of -H, -C _1-6 alkyl, -C(O)R ₂₁ , -CH ₂ OC(O)OR ₂₁ and -CH ₂ OC(O)NR ₂₁ R ₂₂ , -OC(O)OR ₂₁ and -CH ₂ OC(O)NR ₂₁ R ₂₂ ;

wherein R ₂₁ and R ₂₂ are each independently selected from -H and C _1-6 alkyl;

_X7 and _X8 are the same or different, and are independently selected from the group consisting of -O-, _-CH2- , _-CHF- , _-CF2- , -S- and optionally C1- ₃ alkyl substituted -NH-; and

L ₁ is 3 to 6 atoms in length and contains the following structures:

in represents a single bond, a double bond or a triple bond, and wherein (i) occurs 0 or 1 time in L ₁ representing a triple bond or (ii) 0, 1 or 2 occurrences in L ₁ represent a double bond wherein the geometry of each double bond is cis or trans; and (iii) wherein, when 1 occurrence in L ₁ represents a triple bond , 0 occurrences of double bond in L ₁ and (iv) wherein, when 2 occurrences in L ₁ represent double bond , these double bonds are adjacent bonds or alternate bonds;

wherein X ₁₀ , X ₁₁ , X ₁₂ , X ₁₃ , X ₁₄ and X ₁₅ are independently selected from bond; -O-; -NH-; -S-; -C(O)-; -CH-, which are optional is substituted by: (i) Ci _- C3 alkyl, (ii)-OH, or (iii) _-OCH3 ; and _-CH2- , which is optionally substituted by: (i) -OCH3 _; ) C _1-3 alkyl, (ii)-OH, or (iii)-OCH ₃ ;

and any two adjacent members or three adjacent members of a group including X ₁₀ , X ₁₁ , X ₁₂ , X ₁₃ , X ₁₄ and X ₁₅ may optionally form a C _3-6 ring with other atoms Alkyl, optionally substituted with one or more of the following: (i) _C1-3 alkyl, (ii)-OH, or (iii)-OMe; _C5-6 cyclic aryl, any of which optionally substituted with one or more of the following: (i) _C1-3 alkyl, (ii)-OH, or (iii)-OMe; _C5-6 heterocycloalkyl, optionally substituted by a substituted with one or more of the following: (i) _C1-3 alkyl, (ii)-OH, or (iii)-OMe; or a _C3-6 heterocyclic aryl ring, optionally substituted by one or more Substituted with one of the following groups: (i) Ci- _3alkyl , (ii)-OH, or (iii)-OMe; and wherein

B ₁ and B ₂ are independently selected from:

wherein the bonds at points q and r on B ₁ and B ₂ are connected to points q and r on formula (III);

wherein X ₁₆ is selected from the group consisting of -CH ₂ -, -O-, _-NH- , -S- and -C(O)- optionally or -OCH ₃ substituted;

wherein, when present, R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ and R ₁₇ are independently selected from the group consisting of -H, -F, -Cl, -I, -Br, -OH, -SH , -NH ₂ , C _1-3 alkyl, C _3-6 cycloalkyl, -O(C _1-3 alkyl), -O(C _3-6 cycloalkyl), -S(C _1-3 alkyl), -S(C _3-6 cycloalkyl), -NH(C _1-3 alkyl), -NH(C _3-6 cycloalkyl), -N(C _1-3 alkyl) ₂ and -N(C _3-6 cycloalkyl) ₂ ; and

wherein X ₁₇ and X ₁₈ are independently selected from the group consisting of -O-, -CR ₂₃ R ₂₄ -, -NR ₂₃ - and -S-; and wherein

R ₂₃ and R ₂₄ are the same or different, and are selected from the group consisting of -H and -C _1-3 alkyl.

Other embodiments may provide compounds of formula (IV):

or a pharmaceutically acceptable salt thereof, wherein

R _1a is selected from the group consisting of -H, halogen, -C _1-6 alkyl, -N ₃ , -CH ₂ R _2a , -NHR _2a or -OR _2a ;

R _2a is selected from the group consisting of -H and -C _1-6 alkyl;

R _1b is selected from the group consisting of -H, halogen, -C _1-6 alkyl, -OR _2b , -NHR _2b , or with R ₁₁ to form C _4-6 cycloalkyl or C _4-6 hetero cycloalkyl;

R _2b is selected from the group consisting of -H or -C _1-6 alkyl;

R ₃ is -H; -F; -C _1-6 alkyl optionally substituted with: (i) C _1-3 alkyl, (ii) -OH, (iii) halogen, or (iv) )-OCH ₃ ; -C _2-6 alkenyl optionally substituted with: (i) C _1-3 alkyl, (ii)-OH, (iii) halogen, or (iv)-OCH ₃ ; C _2-6 alkynyl optionally substituted with: (i) C _1-3 alkyl, (ii)-OH, (iii) halogen, or (iv)-OCH ₃ ; or optionally -OC _1-6 alkyl substituted with: (i) C _1-3 alkyl, (ii) -OH, (iii) halogen, or (iv) -OCH ₃ ;

R _4a is selected from the group consisting of -H, halogen, -C _1-6 alkyl, -N ₃ , -CH ₂ R _5a , -NHR _5a or -OR _5a ;

R _5a is selected from the group consisting of -H, -C _1-6 alkyl, or R _5a and R ₆ taken together represent -CH ₂ -, -CH ₂ CH ₂ -, -CH(CH ₂ Y ₂ )- or -CH ₂ CH(CH ₂ Y ₂ )-;

Y ₂ is selected from the group consisting of: -H; -OH optionally substituted with -C _1-3 alkyl; -NH ₂ optionally substituted with C _1-3 alkyl; Allyl substituted with: (i)-OH, (ii) _-OCH3 , (iii) Ci- _3alkyl , or (iv) halogen; vinyl optionally substituted with: (i)-OH, (ii) _-OCH3 , (iii) _{Ci_3alkyl} , or (iv) halogen; or _-CH3 optionally substituted with: (i)-OH, ( ii) -OCH ₃ , (iii) C _1-3 alkyl, or (iv) halogen;

R _4b is selected from the group consisting of -H, halogen, -C _1-6 alkyl, -OR _5b , -NHR _5b , or with R ₁₂ to form C _4-6 cycloalkyl or C _4-6 hetero cycloalkyl;

R _5b is selected from the group consisting of -H or -C _1-6 alkyl;

R ₆ is -H; -F; -C _1-6 alkyl optionally substituted with: (i) C _1-3 alkyl, (ii) -OH, (iii) halogen, or (iv) )-OCH ₃ ; -C _2-6 alkenyl optionally substituted with: (i) C _1-3 alkyl, (ii)-OH, (iii) halogen, or (iv)-OCH ₃ ; C _2-6 alkynyl optionally substituted with: (i) C _1-3 alkyl, (ii)-OH, (iii) halogen, or (iv)-OCH ₃ ; or optionally -OC _1-6 alkyl substituted with: (i) C _1-3 alkyl, (ii) -OH, (iii) halogen, or (iv) -OCH ₃ ; or R ₆ is attached to R _5a ;

R ₁₁ is -H or -C _1-6 alkyl, or R ₁₁ and R _1b form a ring;

R ₁₂ is -H or -C _1-6 alkyl, or R ₁₂ and R _4b form a ring;

R ₂₅ and R ₁₈ are the same or different, and are independently selected from the group consisting of -H, C _1-3 alkyl, which is saturated or unsaturated, and optionally by C _1-3 alkyl , -OH, halogen or -OCH ₃ substitution;

Z ₁ and Z ₂ are the same or different, and are independently selected from -O-, -NH-, -N(C _1-3 alkyl), -CH ₂ - and -S-;

P ₁ and P ₂ each independently have an S or R stereochemical configuration;

X _1a and X _2a are the same or different, and are independently selected from O or S;

X _1b and X _2b are the same or different, and are independently selected from the group consisting of -OR ₁₉ , -NR ₁₉ R ₂₀ and -SR ₁₉ -,

wherein R ₁₉ and R ₂₀ are each independently selected from the group consisting of -H, -C _1-6 alkyl, -C(O)R ₂₁ , -CH ₂ OC(O)OR ₂₁ and -CH ₂ OC(O)NR ₂₁ R ₂₂ , -OC(O)OR ₂₁ and -CH ₂ OC(O)NR ₂₁ R ₂₂ ;

wherein R ₂₁ and R ₂₂ are each independently selected from -H and C _1-6 alkyl;

_X7 and _X8 are the same or different, and are independently selected from the group consisting of -O-, _-CH2- , _-CHF- , _-CF2- , -S- and optionally C1- ₃ alkyl substituted -NH-; and

L ₁ is 3 to 6 atoms in length and contains the following structures:

in represents a single bond, a double bond or a triple bond, and wherein (i) occurs 0 or 1 time in L ₁ representing a triple bond or (ii) 0, 1 or 2 occurrences in L ₁ represent a double bond wherein the geometry of each double bond is cis or trans; and (iii) wherein, when 1 occurrence in L ₁ represents a triple bond , 0 occurrences of double bond in L ₁ and (iv) wherein, when 2 occurrences in L ₁ represent double bond , these double bonds are adjacent bonds or alternate bonds;

wherein X ₁₀ , X ₁₁ , X ₁₂ , X ₁₃ , X ₁₄ and X ₁₅ are independently selected from bond; -O-; -NH-; -S-; -C(O)-; -CH-, which are optional is substituted by: (i) Ci _- C3 alkyl, (ii)-OH, or (iii) _-OCH3 ; and _-CH2- , which is optionally substituted by: (i) -OCH3 _; ) C _1-3 alkyl, (ii)-OH, or (iii)-OCH ₃ ;

and any two adjacent members or three adjacent members of a group including X ₁₀ , X ₁₁ , X ₁₂ , X ₁₃ , X ₁₄ and X ₁₅ may optionally form a C _3-6 ring with other atoms Alkyl, optionally substituted with one or more of the following: (i) _C1-3 alkyl, (ii)-OH, or (iii)-OMe; _C5-6 cyclic aryl, any of which optionally substituted with one or more of the following: (i) _C1-3 alkyl, (ii)-OH, or (iii)-OMe; _C5-6 heterocycloalkyl, optionally substituted by a substituted with one or more of the following: (i) _C1-3 alkyl, (ii)-OH, or (iii)-OMe; or a _C3-6 heterocyclic aryl ring, optionally substituted by one or more Substituted with one of the following groups: (i) Ci- _3alkyl , (ii)-OH, or (iii)-OMe; and wherein

B ₁ and B ₂ are independently selected from:

wherein the bonds at points q and r on B ₁ and B ₂ are connected to points q and r on formula (IV);

wherein X ₁₆ is selected from the group consisting of -CH ₂ -, -O-, _-NH- , -S- and -C(O)- optionally or -OCH ₃ substituted;

wherein, when present, R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ and R ₁₇ are independently selected from the group consisting of -H, -F, -Cl, -I, -Br, -OH, -SH , -NH ₂ , C _1-3 alkyl, C _3-6 cycloalkyl, -O(C _1-3 alkyl), -O(C _3-6 cycloalkyl), -S(C _1-3 alkyl), -S(C _3-6 cycloalkyl), -NH(C _1-3 alkyl), -NH(C _3-6 cycloalkyl), -N(C _1-3 alkyl) ₂ and -N(C _3-6 cycloalkyl) ₂ ; and

wherein X ₁₇ and X ₁₈ are independently selected from the group consisting of -O-, -CR ₂₃ R ₂₄ -, -NR ₂₃ - and -S-; and wherein

R ₂₃ and R ₂₄ are the same or different, and are selected from the group consisting of -H and -C _1-3 alkyl.

Other embodiments may provide compounds of formula (V):

or a pharmaceutically acceptable salt thereof, wherein

R _1a is selected from the group consisting of -H, halogen, -C _1-6 alkyl, -N ₃ , -CH ₂ R _2a , -NHR _2a or -OR _2a ;

R _2a is selected from the group consisting of -H and -C _1-6 alkyl;

R _1b is selected from the group consisting of -H, halogen, -C _1-6 alkyl, -OR _2b , -NHR _2b , or with R ₁₁ to form C _4-6 cycloalkyl or C _4-6 hetero cycloalkyl;

R _2b is selected from the group consisting of -H or -C _1-6 alkyl;

R ₃ is -H; -F; -C _1-6 alkyl optionally substituted with: (i) C _1-3 alkyl, (ii) -OH, (iii) halogen, or (iv) )-OCH ₃ ; -C _2-6 alkenyl optionally substituted with: (i) C _1-3 alkyl, (ii)-OH, (iii) halogen, or (iv)-OCH ₃ ; C _2-6 alkynyl optionally substituted with: (i) C _1-3 alkyl, (ii)-OH, (iii) halogen, or (iv)-OCH ₃ ; or optionally -OC _1-6 alkyl substituted with: (i) C _1-3 alkyl, (ii) -OH, (iii) halogen, or (iv) -OCH ₃ ;

R _4a is selected from the group consisting of -H, halogen, -C _1-6 alkyl, -N ₃ , -CH ₂ R _5a , -NHR _5a or -OR _5a ;

R _5a is selected from the group consisting of -H and -C _1-6 alkyl;

R _4b is selected from the group consisting of -H, halogen, -C _1-6 alkyl, -OR _5b and -NHR _5b , or with R ₁₂ forms C _4-6 cycloalkyl or C _4-6 hetero cycloalkyl;

R _5b is selected from the group consisting of -H or -C _1-6 alkyl;

R ₆ is -H; -F; -C _1-6 alkyl optionally substituted with: (i) C _1-3 alkyl, (ii) -OH, (iii) halogen, or (iv) )-OCH ₃ ; -C _2-6 alkenyl optionally substituted with: (i) C _1-3 alkyl, (ii)-OH, (iii) halogen, or (iv)-OCH ₃ ; C _2-6 alkynyl optionally substituted with: (i) C _1-3 alkyl, (ii)-OH, (iii) halogen, or (iv)-OCH ₃ ; or optionally -OC _1-6 alkyl substituted with: (i) C _1-3 alkyl, (ii) -OH, (iii) halogen, or (iv) -OCH ₃ ;

R ₁₁ is -H or -C _1-6 alkyl, or R ₁₁ and R _1b form a ring;

R ₁₂ is -H or -C _1-6 alkyl, or R ₁₂ and R _4b form a ring;

R ₂₅ and R ₁₈ are the same or different, and are independently selected from the group consisting of -H, C _1-3 alkyl, which is saturated or unsaturated, and optionally by C _1-3 alkyl , -OH, halogen or -OCH ₃ substitution;

Z ₁ and Z ₂ are the same or different, and are independently selected from -O-, -NH-, -N(C _1-3 alkyl), -CH ₂ - and -S-;

P ₁ and P ₂ each independently have an S or R stereochemical configuration;

X _1a and X _2a are the same or different, and are independently selected from O or S;

X _1b and X _2b are the same or different, and are independently selected from the group consisting of -OR ₁₉ , -NR ₁₉ R ₂₀ and -SR ₁₉ -,

wherein R ₁₉ and R ₂₀ are each independently selected from the group consisting of -H, -C _1-6 alkyl, -C(O)R ₂₁ , -CH ₂ OC(O)OR ₂₁ and -CH ₂ OC(O)NR ₂₁ R ₂₂ , -OC(O)OR ₂₁ and -CH ₂ OC(O)NR ₂₁ R ₂₂ ;

wherein R ₂₁ and R ₂₂ are each independently selected from -H and C _1-6 alkyl;

_X7 and _X8 are the same or different, and are independently selected from the group consisting of -O-, _-CH2- , _-CHF- , _-CF2- , -S- and optionally C1- ₃ alkyl substituted -NH-; and

L ₁ is 3 to 6 atoms in length and contains the following structures:

in represents a single bond, a double bond or a triple bond, and wherein (i) occurs 0 or 1 time in L ₁ representing a triple bond or (ii) 0, 1 or 2 occurrences in L ₁ represent a double bond wherein the geometry of each double bond is cis or trans; and (iii) wherein, when 1 occurrence in L ₁ represents a triple bond , 0 occurrences of double bond in L ₁ and (iv) wherein, when 2 occurrences in L ₁ represent double bond , these double bonds are adjacent bonds or alternate bonds;

wherein X ₁₀ , X ₁₁ , X ₁₂ , X ₁₃ , X ₁₄ and X ₁₅ are independently selected from bond; -O-; -NH-; -S-; -C(O)-; -CH-, which are optional is substituted with: (i) Ci _- C3 alkyl, (ii) -OH, or (iii) _-OCH3 ; and _-CH2- , which is optionally substituted with: (i) -OCH3 _; ) C _1-3 alkyl, (ii)-OH, or (iii)-OCH ₃ ;

and any two adjacent members or three adjacent members of a group including X ₁₀ , X ₁₁ , X ₁₂ , X ₁₃ , X ₁₄ and X ₁₅ may optionally form a C _3-6 ring with other atoms Alkyl, optionally substituted with one or more of the following: (i) _C1-3 alkyl, (ii)-OH, or (iii)-OMe; _C5-6 cyclic aryl, any of which optionally substituted with one or more of the following: (i) _C1-3 alkyl, (ii)-OH, or (iii)-OMe; _C5-6 heterocycloalkyl, optionally substituted by a substituted with one or more of the following: (i) _C1-3 alkyl, (ii)-OH, or (iii)-OMe; or a _C3-6 heterocyclic aryl ring, optionally substituted by one or more Substituted with one of the following groups: (i) Ci- _3alkyl , (ii)-OH, or (iii)-OMe; and wherein

B ₁ and B ₂ are independently selected from:

wherein the bonds at points q and r on B ₁ and B ₂ are connected to points q and r on formula (V);

wherein X ₁₆ is selected from the group consisting of -CH ₂ -, -O-, _-NH- , -S- and -C(O)- optionally or -OCH ₃ substituted;

wherein, when present, R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ and R ₁₇ are independently selected from the group consisting of -H, -F, -Cl, -I, -Br, -OH, -SH , -NH ₂ , C _1-3 alkyl, C _3-6 cycloalkyl, -O(C _1-3 alkyl), -O(C _3-6 cycloalkyl), -S(C _1-3 alkyl), -S(C _3-6 cycloalkyl), -NH(C _1-3 alkyl), -NH(C _3-6 cycloalkyl), -N(C _1-3 alkyl) ₂ and -N(C _3-6 cycloalkyl) ₂ ; and

wherein X ₁₇ and X ₁₈ are independently selected from the group consisting of -O-, -CR ₂₄ R ₂₃ -, -NR ₂₃ - and -S-; and wherein

R ₂₃ and R ₂₄ are the same or different, and are selected from the group consisting of -H and -C _1-3 alkyl.

Numerous other aspects of embodiments related to Formulas I-V are described throughout this specification. For example, in some embodiments, the pharmaceutically acceptable salt is a diammonium salt. In some embodiments, X _1b and X _2b are -SH. In some embodiments, R _4a is -F, and R _4b , R ₁₂ and R ₆ are -H. In some embodiments, R _4b is -F, and R _4a , R ₁₂ and R ₆ are -H. In other embodiments, R ₁₂ is -F, and R _4a , R _4b and R ₆ are -H. In yet other embodiments, R ₆ is -F, and R _4a , R _4b and R ₁₂ are -H. In yet other embodiments, R _1a is -F, and R ₃ , R _1b and R ₁₁ are -H.

In other embodiments, R ₃ is -F, and R _1a , R _1b and R ₁₁ are -H. In yet other embodiments, R _1b is -F, and R _1a , R ₃ and R ₁₁ are -H. In other embodiments, R ₁₁ is -F, and R _1a , R _1b and R ₃ are -H. In other embodiments, both X _1a and X _2a are =O. In yet other embodiments, B ₁ and B ₂ are both

_In other embodiments, X7 and _X8 are both O. In other embodiments, L ₁ is In yet other embodiments, X _1a is S, X _1b is -SH, X _2a is S, and X _2b is -SH. In still other embodiments, both Z ₁ and Z ₂ are -O-. In other embodiments, X ₁₇ and X ₁₈ are both -O-. In still other embodiments, Z ₁ , Z ₂ , X ₁₇ , and X ₁₈ are each -O-. In yet other embodiments, both R ₂₅ and R ₁₈ are -H.

Embodiments can provide compounds or pharmaceutically acceptable salts having one or more of: (i) an _EC50 value of less than 100 micromolar in a reporter gene assay of cells expressing STING HAQ gene variants; (ii) ) _EC50 values below 100 micromolar in reporter cells expressing human STINGAQ variants; (iii) _EC50 values below 100 micromolar in reporter cells expressing human STINGAQ variants; and (iv) EC50 values in reporter cells expressing human STINGAQ variants Reporter cells for STING REF variants have _EC50 values below 100 micromolar.

Embodiments may provide pharmaceutical compositions comprising a compound or a pharmaceutically acceptable salt described herein and a pharmaceutically acceptable excipient. Embodiments may provide methods of treating cancer comprising administering to a patient an effective amount of a pharmaceutical composition described herein.

Embodiments may further provide mixtures of the compounds described herein, including mixtures of stereoisomers of these compounds. For example, a mixture of Compound 11 and Compound 12 may be provided, or a mixture of Compound 2 and Compound 4 may be provided. Of course, these are not limiting examples and other mixtures are possible.

Specific embodiments are listed in Table 3 below.

table 3

In the above table, compounds 8, 11 and 12 are depicted with the same structural formula and relate to three separate stereoisomers. Applicants note, however, that the phosphochirality of compound 8 is not necessarily the same as that of other compounds labeled "Stereoisomer 1", such as compound 21. The same is true for other stereoisomers.

In some embodiments, the compounds described herein are provided as the free acid. In some embodiments, the compounds are provided as _NH4 salts. In other embodiments, it is a bisTEA salt.

As noted above, embodiments can provide a method of treating cancer in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is administered as the free acid. In some embodiments, the compound is administered as a diammonium salt ( _NH4 ). Pharmaceutical compositions comprising a compound described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient are contemplated. Embodiments may provide compounds of formula I, wherein n is ₁ ; the geometry of the _double bond is trans; X1 and X2 are each SH _; the stereochemistry of P1 is S _; and the stereochemistry of P2 is R; or a pharmaceutically acceptable salt thereof.

Embodiments may provide a method of treating cancer in a patient, the method comprising administering to the patient a compound described herein, or a pharmaceutically acceptable salt or pharmaceutical composition thereof. The cancers treated as described herein can be metastatic cancers. They may be selected from, for example, melanoma, colorectal cancer, breast cancer, acute myeloid leukemia, colon cancer, liver cancer and glioma. Also contemplated are the use of the compounds, salts and pharmaceutical compositions for the treatment of cancer and/or the manufacture of a medicament for the treatment of cancer.

Embodiments may provide a method of treating cancer, the method comprising identifying an individual having a cancer treatable with a compound, pharmaceutically acceptable salt or pharmaceutical composition described herein, and administering to the individual a compound, pharmaceutically acceptable The salt or pharmaceutical composition of which the patient has been identified as treatable with the compound, pharmaceutically acceptable salt or pharmaceutical composition. In some embodiments, an individual is identified as having a cancer treatable with a compound, pharmaceutically acceptable salt, or pharmaceutical composition described herein by the presence of a human REF STING variant allele in the patient.

Embodiments may provide a method of treating cancer in a patient having a REF STING allele, the method comprising administering to the patient a compound described herein, or a pharmaceutically acceptable salt or pharmaceutical composition thereof.

Embodiments may provide a method of treating cancer in a patient having a WT STING allele, the method comprising administering to the patient a compound described herein, or a pharmaceutically acceptable salt or pharmaceutical composition thereof.

Embodiments may provide a method of treating cancer in a patient having an AQ STING allele, the method comprising administering to the patient a compound described herein, or a pharmaceutically acceptable salt or pharmaceutical composition thereof.

Embodiments may provide a method of treating cancer in a patient having a HAQ STING allele, the method comprising administering to the patient a compound described herein, or a pharmaceutically acceptable salt or pharmaceutical composition thereof.

Embodiments may relate to _{C4 - C6 linkers} that may be covalently bound at either end to a purine or pyrimidine base that forms part of a cyclic dinucleotide. In one embodiment, the linker is a butene, pentene or hexene linker bound to a purine base at either end. In another embodiment, the linker is a butene linker bound to a purine base at either end; in another embodiment, the linker is a trans-butene linker with a double bond between the central two carbons.

The following numbered embodiments illustrate the use of these _C4 - _C6 linkers:

1. A compound of formula (X):

or a pharmaceutically acceptable salt thereof, wherein:

_A1 and A2 are sugar moieties and may be the same or different _;

_B3 and _B4 are purine or pyrimidine bases, which may be the same or different, and form nucleotides with A1 and _A2 , respectively _;

L is an alkyl linker;

X ₁₉ and X ₂₀ are the same or different and are selected from the group consisting of -O-, -CH-, -NH- and -S-, wherein -CH- and -NH- may be substituted or unsubstituted replaced.

2. The compound or pharmaceutically acceptable salt according to numbered embodiment 1, wherein -CH- and -NH- of X ₁₉ and X ₂₀ may be substituted with C _1-6 alkyl.

3. The compound or pharmaceutically acceptable salt of numbered embodiment 1, wherein L is butene, pentene or hexane.

4. The compound or pharmaceutically acceptable salt of numbered embodiment 3, wherein L is a double bond centrally located trans-butene linker.

The compounds or pharmaceutically acceptable salts of the numbered embodiments are useful, for example, in the treatment of cancer. A ₁ , A ₂ , B ₃ and B ₄ may be further substituted with, for example, hydroxy, halogen or methoxy. Each phosphorus in formula (X) may be substituted with, for example, -SH, -OH, =O or =S until its valence is satisfied.

Examples of cyclic dinucleotide analogs that may benefit from the linkers of the present invention include, but are not limited to, those identified in: US 2014/0205653 A1; US 2014/0329889 A1; US 2014/0341976 A1; US 2015/ 0056224 A1；US 2016/0362441 A1；US 2017/0158724 A1；US 2017/044206 A1；US 5,547,941；US 7,569,555 B2；US 7,592,326 B2；US 7,709,458 B2；US 9,549,944 B2；WO 2009/133560 A1；WO 2015/074145 A1; WO 2015/077354 A1; WO 2015/185565 A1; WO 2016/100261 A1; WO 2016/120305 A1; WO 2016/145102 A1; WO 2017/093933 A1; WO 2017/123657 A1; WO 2017/175156 A1; EP 1740,192 B1; CN 102199183 A; Corrales, L. et al., “Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic TumorRegression and Immunity, "Cell Reports, 11:1018-1030 (2015); and Lioux, T. et al., "Design, Synthesis, and Biological Evaluation of Novel Cyclic Adenosine-InosineMonophosphate (cAIMP) Analogs That Activate Stimulator of Interferon Genes (STING) ," J. Med. Chem., 59: 10253-10267 (2016). All of these documents, including the compounds therein, are incorporated herein by reference; if any part of any of these documents contradicts or is inconsistent with anything in this specification, the present specification controls .

Description of drawings

Figure 1 shows the synthesis of compound 1a and compound 2a.

Figures 2A and 2B show alternative syntheses of Compound 1 and Compound 1a. This alternative synthesis is also shown in Figure 2C.

FIG. 3 shows the ¹ H NMR spectrum of compound 1. FIG.

Figures 4A, 4B and 4C show the X-ray crystallography results (ORTEP maps) of the asymmetric crystal of Compound 1, the first molecule from the asymmetric crystal, and the second molecule from the asymmetric crystal, respectively.

Figure 4D shows the X-ray crystallography results (ORTEP map) of Compound 2 crystals.

5A and 5B show the synthetic routes of compounds 18, 19 and 20.

Figure 6 shows a map of the expression vector for WT STING (pLenti-WT human STING-Puro).

Figures 7 and 8 accompany Example 108 and show the curative activity of Compound 1a in the CT26 dual tumor model.

Figure 9 accompanies Example 109 and shows tumor volume plots and survival curves for treated tumors.

Figure 10 accompanies Example 110 and shows tumor volume plots and survival curves for treated tumors.

FIG. 11 shows a photograph of the X-ray crystal structure of human WT STING complexed with Compound 1. FIG.

Figure 12 shows the human REF STING C-terminal domain in complex with Compound 1.

FIG. 13 shows synthetic examples of compound 38 and compound 39.

Detailed ways

Provided herein are compounds useful in the treatment of cancer. These compounds activate the stimulator of interferon genes (STING).

In some embodiments, the compound is provided as the free acid. In some embodiments, compounds are provided as, eg, _NH4 salts or TEA salts. Reference to a compound number followed by "a" indicates the diammonium salt of the given compound. For example, "Compound 1a" refers to the diammonium salt of Compound 1.

Embodiments may provide a method of treating cancer in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound of Formula I, Formula II, Formula III, Formula IV, or Formula V, or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is administered as the free acid. In some embodiments, the compound is administered, eg, as a diammonium salt ( _NH4 ). Also provided is a pharmaceutical composition for the treatment of cancer comprising a compound described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

The embodiments described herein can be used to treat cancer or to prepare a medicament for the treatment of cancer. "Cancer" may include, but is not limited to, colon cancer, liver cancer, melanoma, colorectal cancer, breast cancer, acute myeloid leukemia, and glioma.

Those skilled in the art will recognize that when substituents bound to phosphorus atoms (P ₁ , P ₂ ) have both single and double bonds, they may be prone to tautomerism. For example, compounds can tautomerize in equilibrium. An example is shown below:

Such tautomers should be considered within the scope of the claims. The structural representation of any tautomer of a given compound will represent the same compound.

In some embodiments, a compound selected from the group consisting of compounds described herein is provided as the free acid or a pharmaceutically acceptable salt thereof. In some embodiments, the compound selected from the group consisting of the compounds described herein is provided as an _NH4 salt, which may be a diammonium salt.

As used herein, "C _1-6 alkyl" or "C ₁ -C ₆ " alkyl is intended to include C ₁ , C ₂ , C ₃ , C ₄ , C ₅ or C ₆ straight chain (linear) saturated aliphatic _Hydrocarbyl and C3, _C4 , _C5 or _C6 branched saturated aliphatic hydrocarbyl. For example, C _1-6 alkyl is intended to include C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , and C ₆ alkyl. Examples of alkyl groups include moieties having 1 to 6 carbon atoms such as, but not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, sec- Amyl or n-hexyl. Similarly, "C _1-3 alkyl" or "C ₁ -C ₃ alkyl" is intended to include C ₁ , C ₂ or C ₃ straight chain (linear) saturated aliphatic hydrocarbon groups as well as C ₃ branched saturated aliphatic hydrocarbon groups .

As used herein, the term " _{C3-6cycloalkyl} " or " _{C3 - C6cycloalkyl} " _refers to saturated or unsaturated non- Aromatic hydrocarbon ring. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, and cyclohexenyl. The term " _{C5-6cycloalkyl} " or " _C5 -C6cycloalkyl" refers to a saturated or unsaturated non-aromatic hydrocarbon ring having 5 or ₆ carbon atoms (eg, _C5 - _C6 ) .

Unless otherwise specified, the term " _{C5-6heterocycloalkyl} " or " _C5 - _{C6heterocycloalkyl} " refers to a saturated or unsaturated group having one or more heteroatoms (eg, O, N, or S). Saturated non-aromatic 5-6 membered monocyclic group. Unless otherwise specified, the term " _{C4-6heterocycloalkyl} " or " _C4 - _{C6heterocycloalkyl} " refers to a saturated or unsaturated group having one or more heteroatoms (eg, O, N, or S). Saturated non-aromatic 4-6 membered monocyclic group. Examples of heterocycloalkyl include, but are not limited to, piperidinyl, piperazinyl, pyrrolidinyl, dioxanyl, tetrahydrofuranyl, isoindolyl, indoline, imidazolidinyl, pyrazolidine base, oxazolidinyl, isoxazolidinyl, triazolidinyl, oxiranyl, azetidinyl, oxetanyl, thietanyl, 1,2,3 ,6-tetrahydropyridyl, tetrahydropyranyl, tetrahydrothienyl, dihydropyranyl, pyranyl, morpholinyl, 1,4-diazepanyl, 1,4-oxo Azacycloheptanyl, etc.

Other examples of heterocycloalkyl include, but are not limited to, acridinyl, azaoctyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothienyl, benzoxazolyl, benzoxanyl oxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, Carboline, chromanyl, chromenyl, cinnoline, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuranyl , furanyl, furanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolyl, indolyl, indolyl, 3H-indolyl dolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazole base, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl oxazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazol 5(4H)-one, oxazolidinyl, oxazolyl, Oxindolyl, pyrimidinyl, phenanthridine, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl , piperidonyl, 4-piperidinone, piperonyl, pteridyl, purinyl, pyranyl, pyrazinyl, pyrazolidine, pyrazolinyl, pyrazolyl, pyridazinyl, pyridine oxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinoline base, 4H-quinazinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl , 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thienyl, thiazole base, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4- Triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and xanthenyl.

As used herein, the term "C _5-6 aryl" or "C ₅ -C ₆ aryl" refers to 5 to 6 carbon atoms (eg, C ₅ -C ₆ carbon atoms) that do not contain any heteroatoms in the ring structure ) of the aromatic hydrocarbon ring.

Unless otherwise specified, the term "C3-6heteroaryl" or "C3 _{- C6heteroaryl} " refers to an aromatic _3-6 having one or more heteroatoms (eg, O, N, or S). A single-membered monocyclic group, except that the heteroaryl ring will contain no more than one oxygen atom or no more than one sulfur atom.

The term "C _2-6 alkenyl" includes unsaturated aliphatic groups having 2, 3, 4, 5 or 6 carbons and containing at least one double bond. For example, the term "C _2-6 alkenyl" includes straight chain alkenyl groups (eg, vinyl, propenyl, butenyl, pentenyl, hexenyl) and branched alkenyl groups. In certain embodiments, a straight or branched alkenyl group has 6 or fewer carbon atoms in its backbone (eg, C2 _{- C6} straight chain, C3 _{- C6} branched chain). The term "C _2-6 alkenyl" includes alkenyl groups containing 2 to 6 carbon atoms.

The term " _C2-6alkynyl " includes unsaturated aliphatic groups having 2, 3, 4, 5 or 6 carbon atoms and containing at least one triple bond. For example, "alkynyl" includes straight chain alkynyl groups (eg, ethynyl, propynyl, butynyl, pentynyl, hexynyl) and branched alkynyl groups. In certain embodiments, a straight or branched alkynyl group has 6 or fewer carbon atoms in its backbone (eg, C2 _{- C6} straight chain, C3 _{- C6} branched chain). The term "C _2-6 alkynyl" includes alkynyl groups containing 2 to 6 carbon atoms.

treatment method

Embodiments may provide a method of treating cancer in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof.

In some embodiments, the administered compound is provided as the free acid or a pharmaceutically acceptable salt thereof. In some embodiments, the administered compound is provided as an _NH4 salt, the free acid, or a pharmaceutically acceptable salt thereof. In some embodiments, the compounds are provided as _NH4 salts.

The term "optionally substituted" refers to a moiety having a specified substituent that replaces one or more hydrogen atoms on one or more atoms having hydrogen in the moiety.

Chemicals named or depicted are intended to include all naturally occurring isotopes of atoms present in the compounds of the present invention. Isotopes include those atoms with the same atomic number but different mass numbers. By way ^of general example and without limitation, isotopes of1H hydrogen include tritium and deuterium, and isotopes ^of12C carbon ^{include13C and14C} .

dose

The optimal dose for the treatment of cancer can be determined empirically for each individual using known methods, and will depend on a variety of factors, including the activity of the agent; the individual's age, weight, general health, sex and diet; time and route of administration ; and other medications the individual is taking. Optimal dosages can be determined using routine tests and procedures well known in the art. The above compounds can be administered by any suitable route.

As used herein, "pharmaceutically acceptable salts" refer to acid addition or base addition salts of compounds of the present disclosure. A pharmaceutically acceptable salt is any salt that retains the activity of the parent compound and does not produce any undue deleterious or adverse effect on the subject to which it is administered in the environment in which it is administered. Pharmaceutically acceptable salts include, but are not limited to, metal complexes and salts of inorganic acids and carboxylic acids. Pharmaceutically acceptable salts also include metal salts such as aluminum, calcium, iron, magnesium, manganese, and double salts. In addition, pharmaceutically acceptable salts include, but are not limited to, acid salts such as acetate, aspartate, alkylsulfonate, arylsulfonate, axetil, benzenesulfonate acid salt, benzoate, bicarbonate, bisulphate, bitartrate, butyrate, calcium edetate, camphorsulfonate, carbonate, chlorobenzoate, citrate, edetate acid salt, ethanedisulfonate, propionate dodecyl sulfate (estolic), ethanesulfonate (esyl), ethanesulfonate (esylic), formate, fumarate, glucoheptyl gluceptic, gluconate, glutamate, glycolate, glycolylarsanilic, hexamic, hexylresorcinoic ), hydrabamic, hydrobromide, hydrochloride, hydrochloride, hydroiodide, hydroxynaphthoate, isethionate, lactate, lactobionate, Maleate, malate, malonate, mandelate, mesylate, methylnitrate, methylsulfate, mucate, muconic, naphthalenesulfonate ( napsylic), nitrate, oxalate, p-nitromethanesulfonate, palmoate, pantothenate, phosphate, monohydrogen phosphate, dihydrogen phosphate, phthalate, polygalactose Aldehyde, propionate, salicylates, stearate, succinate, sulfamate, sulfanilate, sulfonate, sulfate, tannate, tartrate, tea chloride teoclic, tosylate, etc. Sodium and potassium salts can also be prepared.

Embodiments may be diammonium salts. Pharmaceutically acceptable salts can be derived from amino acids, including but not limited to cysteine. Methods for preparing compounds in salt form are known to those skilled in the art (see, eg, Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH; Verlag Helvetica Chimica Acta, Zurich, 2002; Berge et al. , J. Pharm. Sci. 66:1, 1977).

An "effective amount" of a therapeutic agent is an amount sufficient to provide an observable therapeutic benefit compared to untreated cancer in an individual or patient.

The active agents described herein can be combined with a pharmaceutically acceptable carrier to provide a pharmaceutical formulation thereof. The specific choice of carrier and formulation depends on the particular route of administration for which the composition is intended.

As used herein, "pharmaceutically acceptable carrier" refers to a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the compositions of the present invention include, but are not limited to, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes, disodium hydrogen phosphate, Potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, poly Glycol and Lanolin.

The compositions of the present invention may be suitable for parenteral, oral, inhalation spray, topical, rectal, nasal, buccal, vaginal or implant depot administration, and the like. In some embodiments, the formulation includes ingredients from natural or non-natural sources. In some embodiments, the formulation or carrier can be provided in sterile form. Non-limiting examples of sterile carriers include endotoxin-free or pyrogen-free water.

As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. In certain embodiments, the compound is administered by intravenous, oral, subcutaneous or intramuscular administration. Sterile injectable forms of the compositions of the present invention may be aqueous or oily suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.

For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids and their glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tween, Span, and other emulsifiers commonly used in the preparation of pharmaceutically acceptable solid, liquid or other dosage forms can also be used for formulation purposes.

For oral administration, the compounds or salts may be presented in acceptable oral dosage forms including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, common carriers include lactose and corn starch. Lubricants such as magnesium stearate can also be added. For oral administration in capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral administration, the active ingredient can be combined with emulsifying and suspending agents. Certain sweetening, flavoring or coloring agents may also be added if desired. In addition, preservatives may also be added. Suitable examples of pharmaceutically acceptable preservatives include, but are not limited to, various antibacterial and antifungal agents, such as solvents such as ethanol, propylene glycol, benzyl alcohol, chlorobutanol, quaternary ammonium salts, and parabens ( For example, methylparaben, ethylparaben, propylparaben, etc.).

"Immediate release" is intended to include conventional release, wherein the release of the drug begins immediately after administration. As used herein, the term "immediate release" includes dosage forms that allow dissolution of the drug in the gastrointestinal contents, with no intention of delaying or prolonging the dissolution or absorption of the drug. The goal is to release the drug quickly after administration, eg, release at least 80% of the drug within approximately 30 minutes after onset of dissolution in a dissolution test.

"Sustained release" or "extended release" includes dosage forms in which the time course and/or location characteristics of drug release are selected to achieve therapeutic or convenience purposes not provided by conventional dosage forms such as solutions or immediate release dosage forms.

The term "steady state" means that plasma levels of a given active agent have been reached and subsequent doses of active agent remain at or above the minimum effective therapeutic level for the given active agent and below the minimum toxic plasma level for the given active agent s level.

As used herein, the term "single formulation" refers to a single carrier or vehicle formulated to deliver effective amounts of both therapeutic agents to a patient. A single vehicle is designed to deliver an effective amount of each agent along with any pharmaceutically acceptable carrier or excipient. In some embodiments, the vehicle is a tablet, capsule, pill, or patch.

The term "unit dose" is used herein to refer to the simultaneous administration of two agents in one dosage form to a patient being treated. In some embodiments, the unit dose is a single formulation. In certain embodiments, a unit dose includes one or more vehicles, such that each vehicle contains an effective amount of the agent together with pharmaceutically acceptable carriers and excipients. In some embodiments, a unit dose is one or more tablets, capsules, pills or patches administered to a patient simultaneously.

As used herein, the term "dosage range" refers to the upper and lower limits of acceptable variation in a given pharmaceutical dose. In general, any amount of the agent dosage within the specified range can be administered to a patient being treated.

The term "treating" is used herein to refer to alleviating, reducing or alleviating at least one symptom of a disease in an individual. For example, with respect to cancer, the term "treating" can refer to retarding, delaying the onset (ie, the period preceding the clinical manifestation of the disease or disease symptoms), and/or reducing the risk of developing or worsening disease symptoms. The term "protect" is used herein to refer to preventing, delaying or treating (or all, as the case may be) the development or persistence or exacerbation of disease symptoms in an individual.

The terms "individual" or "patient" are intended to include animals capable of developing or afflicted by cancer. Examples of individuals or patients include mammals such as humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In certain embodiments, the individual is a human, eg, a human suffering from, at risk of, or potentially capable of developing cancer.

The terms "about" or "approximately" generally mean within 20% of a given value or range, more preferably within 10%, and most preferably still within 5%. Alternatively, particularly in biological systems, the term "about" means approximately within a logarithm (ie, an order of magnitude), preferably within twice a given value.

In the context of describing the invention (especially in the context of the claims below), the terms "a (a)" and "an (an)" and "the (the) are used unless otherwise indicated herein or otherwise clearly contradicted by context. )" and similar referents should be understood to encompass both the singular and the plural. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (ie, meaning "including, but not limited to,") unless otherwise specified. Unless otherwise indicated herein, ranges of values recited herein are merely intended as a shorthand way of individually representing each separate value within the range, and each separate value is incorporated into this specification as if it were individually as described.

Exemplary cell proliferative disorders that can be treated using one or more of the compounds disclosed herein include, but are not limited to, cancer, precancerous or precancerous conditions, and metastatic lesions in tissues and organs in the body. Cell proliferative disorders can include hyperplasia, metaplasia, and dysplasia.

The compounds disclosed herein, or pharmaceutically acceptable salts thereof, are useful in the treatment or prevention of cell proliferative disorders, or in individuals at increased risk of developing cancer relative to most populations, for the treatment or prevention of cancer, or for these Aim to identify suitable candidates.

Pharmaceutical Formulations and Routes of Administration

Provided herein are pharmaceutical formulations comprising a compound or a pharmaceutically acceptable salt thereof for use in the treatment of cancer. The pharmaceutical preparations may additionally contain carriers or excipients, stabilizers, flavoring and/or coloring agents.

The compound, or a pharmaceutically acceptable salt thereof, can be administered using a variety of routes of administration known to those skilled in the art. Routes of administration include oral administration. In certain embodiments, a pharmaceutical formulation comprising a compound or a pharmaceutically acceptable salt thereof can be administered orally in the form of a liquid, syrup, tablet, capsule, powder, spray, chewable tablet or dissolvable wafer. Alternatively, the pharmaceutical formulations of the present invention may be administered intravenously or transdermally. Other routes of administration are known to those of skill in the art (see, eg, Remington's Pharmaceutical Sciences, Ed. Gennaro A.R., 20th Edition, Mack Publishing Co., Easton, Pa.).

In some embodiments, the compounds or pharmaceutically acceptable salts are formulated as pastes, jellies or suspensions. For example, the drug is dissolved, entrapped or suspended in a gelatinous solution or semi-solid in the form of drug particles, microencapsulated particles or drug-polymer particles. One advantage of oral jelly formulations is that it is easier to administer the drug to patients who have difficulty swallowing tablets, capsules or pills. In certain embodiments, the compounds are mixed well and suspended in a suitable medium to form a paste or gel. Other agents may optionally be admixed to provide flavor during oral administration. Peanut butter or alginate flavored with raspberries and sweeteners are many examples of suitable taste masking agents. In various embodiments, pastes or jellies can also be formulated with suitable binders or excipients known in the art for topical administration.

Methods of preparing sustained release formulations in the form of tablets, capsules or pills are known in the art. In some embodiments, sustained release formulations are prepared by coating the active ingredient of the drug with a polymer, preferably a water-insoluble polymer. For example, a water-insoluble polymer used as a sustained-release coating agent, enteric coating agent or gastric coating agent in the pharmaceutical field. Water insoluble polymers may include, for example, ethyl cellulose, purified shellac, white shellac, aminoalkyl methacrylate copolymer RS, hydroxypropyl methylcellulose phthalate, hydroxypropylmethyl acetate succinate cellulose, carboxymethyl ethyl cellulose, cellulose acetate phthalate, methacrylic acid copolymer L, methacrylic acid copolymer LD, methacrylic acid copolymer S, methacrylic acid aminoalkyl ester copolymer E or polyvinyl acetal diethylaminoacetate.

The type, degree of substitution, and molecular weight of the water-insoluble polymer may depend on the solubility of the active ingredient in water or alcohol, the level of sustained release desired, and the like. The water-insoluble polymers may be used alone or in combination. Hydrogenated oils, stearic acid or cetyl alcohol can also be incorporated as coating aids, and medium chain triglycerides, triacetin, triethyl citrate or cetyl alcohol as plasticizers.

In some embodiments, the sustained release formulation is a matrix-type tablet or granule. The active ingredient can be coated with up to three different types of polymers. These three different types of polymers can include: 1) water insoluble polymers such as ethyl cellulose; 2) pH independent gelling polymers such as hydroxypropyl methyl cellulose; and 3) pH dependent polymers pH gelling polymers such as sodium alginate. These three different types of polymers can be used together to reduce the release rate of the drug.

Dosage Form: Release Properties

Sustained release formulations can achieve a certain degree of sustained effect. However, the exposure and/or bioavailability of an active ingredient can vary based on a variety of factors, such as the absorption window, the carrier or excipient used in the formulation, the mode of delivery of the formulation, and/or the transport of the active ingredient through the patient's gastrointestinal tract time.

The therapeutic agent may contain at least one sustained release moiety for performing a sustained release function and an immediate release moiety for performing an immediate release function. In certain embodiments, when the therapeutic agent is in a single dosage form, it can be in the form of a tablet formed from a mixture of sustained-release particles that make up the sustained-release portion and immediate-release particles that make up the immediate-release portion; A capsule preparation obtained by filling a capsule with granules and immediate-release granules, or a compression-coated tablet in which an outer layer constituting an immediate-release portion is formed on an inner core constituting a sustained-release portion. However, there is no limitation to the above-described embodiments.

In addition, there is no particular limitation on the accommodating state of the drug in the composition or the immediate-release portion or the sustained-release portion; the compound may be uniformly dispersed in the composition, the immediate-release portion or the sustained-release portion, or may be contained only in the composition, the immediate-release portion A portion or a portion of a sustained release portion, or may be contained with a concentration gradient.

The sustained release portion of the composition according to the present invention may contain at least one pH-independent polymeric substance or pH-dependent polymeric substance for controlling drug release.

As used herein, pH-independent polymeric species may include polymeric species whose charge state is generally found in the gastrointestinal tract under pH conditions, particularly pH 1 to pH 8, which are nearly constant. This means, for example, that the polymeric species does not have functional groups whose charge state varies with pH, such as basic functional groups such as amino groups or acidic functional groups such as carboxylic acid groups. Note that the pH-independent polymeric substance may be included for the purpose of imparting a sustained release function to the composition according to the present invention, but may also be included for another purpose. Furthermore, the pH-independent polymeric species used in the present invention may be insoluble in water, or may swell or dissolve in water to form a gel.

Examples of water-insoluble pH-independent polymeric species include, but are not limited to, cellulose ethers, cellulose esters, and methacrylic acid-acrylic acid copolymers (trade name Eudragit, manufactured by Rohm GmbH & Co. KG, Darmstadt, Germany). Examples include, but are not limited to, cellulose alkyl ethers, such as ethyl cellulose (trade name Ethocel, manufactured by Dow Chemical Company, USA), ethyl methyl cellulose, ethyl propyl cellulose, or isopropyl cellulose, and butyl cellulose cellulose; aralkyl ethers of cellulose, such as benzyl cellulose; cyanoalkyl ethers of cellulose, such as cyanoethyl cellulose; organic acid esters of cellulose, such as cellulose acetate butyrate, cellulose acetate, propionic acid Cellulose or cellulose butyrate, and cellulose acetate propionate; ethyl acrylate-methyl methacrylate copolymer (trade name Eudragit NE, manufactured by Rohm GmbH & Co. KG, Darmstadt, Germany) and aminoalkyl methacrylate Ester Copolymer RS (tradename Eudragit RL, Eudragit RS). The average particle size of the water-insoluble polymer used in the present invention is not particularly limited, but generally the smaller the average particle size, the better the performance, and the average particle size is preferably 0.1-100 μm, more preferably 1-50 μm, particularly preferably 3-15 μm, most preferably 5-15 μm. In addition, examples of water-soluble or water-swellable pH-independent polymeric substances include, but are not limited to, polyethylene oxide (trade name Polyox, manufactured by The Dow Chemical Company, molecular weight 100,000 to 7,000,000), low-substituted hydroxypropyl Base cellulose (trade name L-HPC, manufactured by Shin-Etsu Chemical, Japan), hydroxypropyl cellulose (trade name HPC, manufactured by NipponSoda, Co., Ltd, Japan), hydroxypropyl methylcellulose ( Trade names Metolose 60SH, 65SH, 90SH, manufactured by Shin-Etsu Chemical, Japan) and methylcellulose (trade name Metolose SM, manufactured by Shin-Etsu Chemical, Japan).

In some embodiments, the composition may contain a single pH-independent polymeric species, or may contain multiple pH-independent polymeric species. If used in the embodiments described herein, the pH-independent polymeric material may be a water-insoluble polymeric material, more preferably ethyl cellulose, ethyl acrylate-methyl methacrylate copolymer (trade name Eudragit NE). ) or aminoalkyl methacrylate copolymer RS (trade name Eudragit RL, Eudragit RS). At least one of ethyl cellulose and aminoalkyl methacrylate copolymer RS is particularly preferred. Most preferred is ethyl cellulose. The amount of the pH-independent polymer substance contained in the composition is not particularly limited; the amount can be appropriately adjusted according to the purpose of, for example, controlling sustained release of the drug.

The pH-dependent polymeric species useful in the embodiments described herein may be polymeric species that vary in charge state at pH conditions commonly found in the gastrointestinal tract, particularly pH 1 to pH 8. This means, for example, that the polymeric species has functional groups whose charge state varies with pH, for example basic functional groups such as amino groups or acidic functional groups such as carboxylic acid groups. The pH-dependent functional groups of the pH-dependent polymer species are preferably acidic functional groups, and the pH-dependent polymer species most preferably have carboxylic acid groups.

The pH-dependent polymeric species used in the present invention may be insoluble in water, or may swell or dissolve in water to form a gel. Examples of pH-dependent polymeric materials for use in the present invention include, but are not limited to, enteric polymeric materials. Examples of enteric polymeric substances include, but are not limited to, methacrylic acid-methyl methacrylate copolymer (Eudragit L100, Eudragit S100, manufactured by Rohm GmbH & Co. KG, Darmstadt, Germany), methacrylic acid-ethyl acrylate copolymer (Eudragit L100-55, Eudragit L30D-55, manufactured by Rohm GmbH & Co. KG, Darmstadt, Germany), hydroxypropyl methylcellulose phthalate (HP-55, HP-50, manufactured by Shin-Etsu Chemical, Japan ), hydroxypropyl methylcellulose acetate succinate (AQOAT, manufactured by Shin-Etsu Chemical, Japan), carboxymethyl ethyl cellulose (CMEC, manufactured by Freund Corporation, Japan), and cellulose acetate phthalate .

Examples of pH-dependent polymeric species that swell or dissolve in water to form gels include, but are not limited to, alginic acid, pectin, carboxyvinyl polymers, and carboxymethyl cellulose. In the present invention, a single pH-dependent polymeric species may be contained in the composition, or multiple pH-dependent polymeric species may be contained. The pH-dependent polymer substance used in the present invention is preferably an enteric polymer substance, more preferably methacrylic acid-ethyl acrylate copolymer, methacrylic acid-methyl methacrylate copolymer, hydroxypropyl phthalate methyl cellulose or hydroxypropyl methyl cellulose acetate succinate, particularly preferably a methacrylic acid-ethyl acrylate copolymer.

When the pH-dependent polymer substance is used in the manufacturing process of the composition according to the present invention, a commercially available product in powder or granular form can be used as it is, or in which the pH-dependent polymer substance has been dispersed in a solvent in advance The suspension type product, or this commercially available product can be dispersed in water or organic solvent. The smaller the particle size of the pH-dependent polymer substance, the better the performance, and the pH-dependent polymer substance is preferably a powder type. In the case of a methacrylic acid-ethyl acrylate copolymer, an example is Eudragit L100-55. The average particle size of the pH-dependent polymer substance used in the present invention is not particularly limited, but the average particle size is preferably 0.05-100 μm, more preferably 0.05-70 μm, and most preferably 0.05-50 μm. In addition, the amount of the pH-dependent polymer substance is not particularly limited, for example, in the case of an enteric polymer substance, the amount is usually 0.1-90 parts by weight, preferably 1-70 parts by weight based on 100 parts by weight of the composition parts by weight, more preferably 5-60 parts by weight, particularly preferably 10-50 parts by weight.

Therapeutic agents according to the embodiments described herein may also contain any of various additives, such as any of various pharmacologically acceptable carriers such as diluents, lubricants, binders and disintegrants, and preservatives, as desired , colorants, sweeteners, plasticizers, film coating agents, etc. Examples of diluents include, but are not limited to, lactose, mannitol, calcium hydrogen phosphate, starch, pregelatinized starch, crystalline cellulose, light silicic anhydride, synthetic aluminum silicate, magnesium aluminum metasilicate, and the like. Examples of lubricants include, but are not limited to, magnesium stearate, calcium stearate, talc, sodium stearoyl fumarate, and the like. Examples of binders include, but are not limited to, hydroxypropylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and the like. Examples of disintegrants include, but are not limited to, carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, and the like. Examples of preservatives include, but are not limited to, parabens, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, and the like. Preferred examples of colorants include, but are not limited to, water-insoluble lake pigments, natural pigments (eg, beta-carotene, chlorophyll, red iron oxide), yellow iron oxide, red iron oxide, black iron oxide, and the like. Preferred examples of sweeteners include, but are not limited to, sodium saccharin, dipotassium glycyrrhizinate, aspartame, stevia, and the like. Examples of plasticizers include, but are not limited to, glycerol fatty acid esters, triethyl citrate, propylene glycol, polyethylene glycol, and the like. Examples of film coating agents include, but are not limited to, hydroxypropyl methylcellulose, hydroxypropyl cellulose, and the like.

Manufacturing method

To make the embodiments described herein, a single conventional method or a combination of conventional methods can be used. For example, when making drug-containing granules as a sustained release part or an immediate release part, granulation is the main operation, but this operation can be combined with other operations such as mixing, drying, sieving and sorting. As the granulation method, there can be used, for example, a wet granulation method in which a binder and a solvent are added to powder and granulated, a dry granulation method in which powder is compressed and granulated, a binder heated and melted and heated and granulated can be used. Melt granulation method of granulation, etc.

In addition, depending on the granulation method, operation methods such as: a mixing granulation method using a planetary mixer, a screw mixer, etc., a high-speed mixing granulation method using a Henschel mixer, a Super mixer, etc., a cylindrical mixer, etc. can be used. Extrusion granulation method, wet high shear granulation method, fluidized bed granulation method, compression granulation method, etc. Pulverization granulation method or spray granulation method. After granulation, it can be dried using a dryer, fluidized bed, etc., pulverized and sieved to obtain granules or fine particles for use. Furthermore, granulation solvents may be used when preparing the compositions according to the present invention. This granulation solvent is not particularly limited, and the granulation solvent may be water or any one of various organic solvents, such as water, lower alcohols such as methanol or ethanol, ketones such as acetone or methyl ethyl ketone, dichloride Methane or mixtures thereof.

For the sustained release particles contained in the embodiments, at least one drug and at least one polymeric substance selected from the group consisting of pH-independent polymeric substances and pH-dependent polymeric substances are mixed together, and diluted as needed agent and binder, and granulated to obtain granular material. The obtained particulate matter is dried using a chamber dryer, fluidized bed dryer, etc., and sieved using a grinder or a shaker, whereby sustained-release particles can be obtained. Alternatively, as a method for producing sustained-release granules in the present invention, a dry compactor such as a roller press or a slugtabletting machine may be used to add at least one drug, at least one selected from pH-independent The polymer substance and the polymer substance of the pH-dependent polymer substance, as well as diluents and binders are added as required, and are subjected to compression molding while mixing, and then granulated by pulverizing to a suitable size. The particulate matter prepared using such a granulator can be used as such as granules or fine particles according to the present invention, or can be further pulverized using a power mill, roll granulator, rotor speed mill, etc., and sieved for sustained release granules. Note that immediate release particles can also be produced as sustained release particles.

Compression-molded products can be manufactured using a single conventional method or a combination of conventional methods to produce a drug-containing sustained-release portion or an immediate-release portion, or the compositions described herein. For example, at least one drug, at least one polymeric substance selected from the group consisting of pH-independent polymeric substances and pH-dependent polymeric substances, diluents such as mannitol or lactose, binders such as polyvinylpyrrolidone or Crystalline cellulose, a disintegrant such as sodium carboxymethyl cellulose or crospovidone, and a lubricant such as magnesium stearate or talc, and tableting using an ordinary method, whereby a compression-molded product can be obtained. In this case, tableting is the main operation in the process of making compression-molded products, but this operation can be combined with other operations such as mixing, drying, sugar coating and coating.

Examples of tablet compression methods include, but are not limited to, direct compression molding, in which at least one drug and a pharmacologically acceptable additive are mixed together, and the mixture is then compressed directly into tablets using a tablet machine, and dry granule compression or Wet granulation compression, wherein sustained release granules or immediate release granules according to the present invention are compression molded after adding lubricants or disintegrants as required. The tableting machine used for compression molding is not particularly limited; for example, a single-punch tableting machine, a rotary tableting machine, or a press-coated tabletting machine can be used.

Drug-containing sustained-release granules or immediate-release granules, or compression-molded products according to embodiments herein may be used as compositions in the form of granules or tablets, but may also be further processed to manufacture compositions. For example, compression molded products or granules can use film based materials such as ethyl cellulose, casein, methyl cellulose, hydroxypropyl methyl cellulose, methacrylic acid copolymer L, cellulose acetate phthalate, insect A film-coated tablet or a sugar-coated tablet is prepared by applying a film coating with a gum or the like, or applying a sugar coating using a sugar coating liquid containing sucrose, sugar alcohol, gum arabic powder, talc, and the like. One solvent in this coating technique can be purified water, but organic solvents such as alcohols, ketones, ethers or chlorinated hydrocarbons, or mixtures thereof, can also be used. For example, ethanol, acetone, dichloromethane, etc. can be used as the organic solvent. In addition, as the coating apparatus, those commonly used in coating techniques for manufacturing pharmaceuticals can be used, and examples include a spray coating apparatus in which coating is performed by spraying a coating liquid or the like, and a rotor fluidized bed for layering Granulator.

In the case of manufacturing a capsule formulation, the capsule formulation can be manufactured by filling sustained-release granules or immediate-release granules or microtablets as described above into hard gelatin capsules or HPMC capsules using an automatic capsule filling machine. Alternatively, in the case of a tube-administered preparation or a dry syrup used in admixture with water or the like at the time of administration, sustained-release granules or immediate-release granules as described above may be mixed with a thickening or dispersing agent to disperse these granules, and then The mixture is made into granules or tablets. In addition, a liquid or jelly can be prepared using water and a substance selected from the group consisting of dispersing agents, emulsifiers, thickening agents, preservatives, pH adjusters, sweeteners, flavoring agents, aromatic agents, and the like. However, regarding other manufacturing methods, there is no limitation to the above.

For a more complete understanding of the embodiments described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting.

Example

The following abbreviations may be used in all examples.

All: allyl

BOP: (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate

DMT: 4,4′-dimethoxytrityl

(DMTO-:

Bz: Benzoyl

ib: isobutyryl

Hunig base: i-Pr ₂ NEt (diisopropylethylamine)

AllylOH: allyl alcohol

OAll: -OCH ₂ CHCH ₂

ACN: Acetonitrile

All: -CH ₂ CHCH ₂

2-NitroBnBr: 2-nitrobenzyl bromide

Bz: Benzoyl

ib: isobutyryl

i-Pr: isopropyl

CE: cyanoethyl

DEAD: Diethyl azodicarboxylate

DIAD: Diisopropyl azodicarboxylate

DCM: dichloromethane

DDTT: N,N-Dimethyl-N'-(3-thione-3H-1,2,4-dithiazol-5-yl)formamidine

DMOCP: 2-Chloro-5,5-dimethyl-1,3,2-dioxaphosphine 2-oxide

TBS: tert-butyldimethylsilyl

3H-benzo[c][1,2]dithiol-3-one:

Example 1--Synthesis of Compound 1a

The complete scheme of this synthesis is available in Figure 1.

Step A

To (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)( Phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (Compound 100) (mixture of phosphorus diastereomers; 80.0 g , 91.332 mmol, 1 equiv, ChemGenes catalog #ANP-9151), allyl alcohol (9.63 ml, 142 mmol, 1.55 equiv) and triphenylphosphine (38.3 g, 146 mmol, 1.60 equiv) in THF (1.1 L) To the mixture was added DEAD (40 wt% solution in toluene; 54.2 ml, 137 mmol, 1.5 equiv). Stirring was continued at ambient temperature and the reaction was monitored by LC/MS. Upon completion (19 hours), the mixture was concentrated in vacuo (35°C) and the resulting mixture was subjected to silica gel column chromatography (800 g x 2 columns, 40-60% EtOAc in n-heptane buffered with 0.5% triethylamine) Purification gave compound 101 as a white foam (84.2 g, quantitative yield, mixture of phosphorus diastereomers).

¹ H NMR (3:2 mixture of phosphorus diastereomers, 400 MHz, CDCl ₃ ) 1.14-1.21 (m, 12H) 2.40 (t, J=6.2 Hz, 1.2H) 2.59 (t, J=6.2 Hz) ,0.8H)3.27(d,J=8.6Hz,1H)3.52-3.66(m,5H)3.78(s2.4H)3.79(s 3.6H)4.28-4.34(m,1H)4.84-4.96(m,0.4 H)4.99(d,J＝5.5Hz,2H)4.95-5.10(m,0.6H)5.05(d,J＝10.9Hz,1H)5.22(br d,J＝17.6Hz,1H)5.64(br d, J=53.2Hz,0.6H)5.70(br d,J=51.6Hz,0.4H)5.96-6.75(m,1H)6.20(d,J=16.0Hz,0.6H)6.24(d,J=17.2Hz, 0.4H)6.74-6.79(m,4H)7.02-7.06(m,2H)7.17-7.24(m,8H)7.32-7.34(m,2H)7.41-7.44(m,2H)8.11(s,1H)8.52 (s, 0.4H) 8.54 (s, 0.6H).

Step B

To a solution of compound 101 (3.00 g, 3.28 mmol, 1 equiv) in acetonitrile (30 ml) was added water (0.118 ml, 6.55 mmol, 2.0 equiv) and pyridine trifluoroacetate (0.759 g, 3.93 mmol, 1.2 equiv) ). After stirring for 1 min at ambient temperature, tert-butylamine (14.5 g, 21.0 ml, 0.20 mol, 60 equiv) was added. After complete cleavage of the cyanoethyl group (monitored by LC/MS), the reaction mixture was concentrated in vacuo and azeotroped twice with acetonitrile. The crude mixture was dissolved in DCM (45.0 ml) and treated with water (0.118 ml, 6.55 mmol, 2.0 equiv) and _NaHSO4 - _SiO2 (1.18 g, 6.55 mmol, 2 equiv) at ambient temperature. After complete cleavage of the DMT group (monitored by LC/MS, about 1 hour), the reaction mixture was filtered and rinsed twice with DCM/MeOH (9/1, 20 ml). The combined filtrates were concentrated in vacuo and treated with a 1:1 mixture of n-heptane/toluene (about 30 ml). The top layer was removed by decantation. The same procedure was repeated again with n-heptane/toluene (1/1, 30 ml) and the bottom layer was azeotroped twice with acetonitrile to give compound 102 (assuming 100% theoretical yield). The product was used in the next step without further purification.

step C

To a mixture of compound 102 (1.56 g, 3.27 mmol, 1 equiv) and compound 101 (3.00 g, 3.28 mmol, 1 equiv) in acetonitrile (30 ml) was added pyridine trifluoroacetate (azeotropically dried with pyridine; 0.760 g, 3.94 mmol, 1.25 equiv). After 5 minutes, DDTT (0.840 g, 4.09 mmol, 1.30 equiv, ChemGenes catalog #RN-1588) was added, and after complete sulfurization (monitored by LC/MS), the reaction mixture was concentrated in vacuo. The residue was dissolved in DCM (30 ml) and treated in DCM (30 ml) with water (0.57 ml, 32 mmol, 10 equiv) and 6% dichloroacetic acid in DCM (1.56 ml, 18.9 mmol, 6.0 equiv). After 20 minutes, the reaction was quenched with pyridine (20 mL) and concentrated in vacuo. The residue was azeotroped with pyridine to give compound 103 (3.22 g, assuming 100% theoretical yield). The product was used in the next step without further purification.

step D

To a solution of compound 103 (3.22 g, 3.15 mmol, 1 equiv) in pyridine (100 ml) was added DMOCP (1.45 g, 7.88 mmol, 2.50 equiv) at ambient temperature. After complete macrocyclization (monitored by LC/MS), water (1.7 ml, 94.5 mmol, x 10 times relative to DMOCP) was added, followed by 3H-benzo[c][1,2]dithiol-3 - Ketone (0.795 g, 4.73 mmol, 1.5 equiv). After complete sulfidation (about 40 minutes), the reaction mixture was partially concentrated in vacuo to about 15 ml and poured into a mixture of saturated aqueous _NaHCO3 (50 ml) and water (30 ml). After stirring for 10 minutes at ambient temperature, the mixture was extracted with a 1:1 mixture of EtOAc/MTBE (60 ml x 3 times). The organic layers were combined, washed with brine (25ml), dried _{over Mg2SO4} , and concentrated in vacuo. The residue was purified by silica gel column chromatography (0-20% MeOH in DCM) to give compound 104 (3.31 g, 3.20 mmol, assuming 100% theoretical yield) as a brown oil. The product was used in the next step without further purification.

step E

To a solution of compound 104 (3.31 g, 3.20 mmol, 1 equiv) in acetonitrile (66.2 ml) was added 2-nitrobenzyl bromide (2.42 g, 11.2 mmol, 3.50 equiv) and triethylamine (1.78 ml, 12.8 mmol, 4.00 equiv). After complete reaction (monitored by LC/MS, about 20 hours at ambient temperature), the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography (60% ethyl acetate/n-heptane to 100% ethyl acetate) to give 0.568 g of product as a mixture of phosphorus diastereomers. Preparative HPLC separation of the diastereomers afforded compound 105 (SR isomer; 0.225 g, 0.180 mmol, 5.6% overall yield from compound 101) and compound 106 (RR isomer; 0.187 g, 0.149 mmol, the overall yield from compound 1 is 4.7%).

Compound 105 (SpRp) ¹ H NMR (400 MHz, CDCl ₃ ) δ=8.63 (s, 1H), δ=8.61 (s, 1H), 8.04-8.00 (m, 2H), 7.99 (s, 1H), 7.90 ( s,1H),7.65-7.44(m,8H),7.40-7.31(m,4H),7.25-7.21(m,4H),6.15-5.89(m,5H),5.61(dd,J=52.0,5.1 Hz,1H),5.55(ddd,J=51.2,4.7,2.7Hz,1H)5.51-5.42(m,1H),5.31-5.22(m,2H),5.11(dd,J=3.9,9.8Hz,2H ),5.04-4.95(m,4H),4.55-4.37(m,7H),4.29-4.12(m,3H)

Compound 106 (RpRp) ¹ H NMR (400 MHz, CDCl ₃ ) δ=8.65 (s, 2H), 8.06 (dd, J=1.4, 8.0 Hz, 2H), 7.98 (s, 2H), 7.57-7.52 (m, 6H), 7.47-7.32(m, 6H), 7.25-7.21(m, 4H), 6.15(d, J=18.7Hz, 2H), 6.09-5.99(m, 2H), 5.82-5.76(m, 2H) ,5.60(dd,J＝51.8,4.9Hz,2H),5.27(dd,J＝1.2,17.2Hz,2H),5.12(dd,J＝1.0,10.4Hz,2H),5.06-4.96(m,4H ),4.55-4.40(m,4H),4.36-4.24(m,4H),4.21-4.02(m,2H)

Preparative HPLC conditions:

Step F

To a heated (90°C) solution of compound 105 (519 mg, 0.414 mmol, 1 equiv) in toluene (519 ml) was added Hoveyda-Grubbs Catalyst ^™ of the second generation ((1,3-bis-(2,4,6- Trimethylphenyl)-2-imidazolidineidene)dichloro(o-isopropoxyphenylmethylene)ruthenium; can Catalog No. 569755 obtained; CAS301224-40-8; 91 mg, 0.15 mmol, 0.35 equiv) and quinone (0.102 ml, 1.243 mmol, 3.0 equiv). The mixture was heated to reflux and the progress of the reaction was monitored by LC/MS. After 3 hours, additional catalyst (91 mg, 0.15 mmol, 0.35 equiv) was added and the reaction continued for an additional 3 hours. After cooling, the mixture was treated with DMSO (0.59 ml, 8.3 mmol, 20 equiv) at ambient temperature for 15 h, concentrated in vacuo, and subjected to silica gel column chromatography ( _SiO2 25 g, 66% ethyl acetate in n-heptane to 100% ethyl acetate) to give compound 107 as a brown dry foam (200 mg, 0.163 mmol, 39% yield).

¹ H NMR (400MHz, CDCl ₃ ) δ=8.19(s, 1H), 8.12(dd, J=7.8Hz, 1.9Hz, 1H), 8.10(s, 1H), 8.02(d, J=8.2Hz, 1H ),7.89(s,1H),7.63(br d,J=7.0Hz,1H),7.53-7.41(m,10H),7.35-7.30(m,2H),7.25-7.20(m,4H),6.23 (d, J=17.6Hz, 1H), 6.14 (d, J=18.8Hz, 1H), 5.86-5.75 (m, 1H), 5.75 (dt, J=15.3, 5.0Hz, 1H), 5.67 (dt, J=15.3, 4.7Hz, 1H), 5.60 (dd, J=52.0, 3.9Hz, 1H), 5.48 (dd, J=50.4, 3.9Hz, 1H), 5.50-5.39 (m, 1H), 4.91-4.64 (m, 4H), 4.57-4.25 (m, 9H), 4.15 (d, J=7.03 Hz, 1H), 4.11 (d, J=7.03 Hz, 1H).

step G

To a solution of compound 107 (88 mg, 0.072 mmol, 1 equiv) in 1,4-dioxane (1.76 ml) was added thiophenol (0.88 mL, 8.55 mmol, 119 equiv) and triethylamine (0.88 mL, 6.31 mmol, 88 equiv). The resulting mixture was stirred at ambient temperature. After complete reaction (monitored by LC/MS, 13 hours), methanol (5.28 ml) and 28% ammonium hydroxide (3.52 ml) were added and the resulting mixture was heated to 50°C. After complete reaction (monitored by LC/MS, 5 hours), the mixture was cooled to ambient temperature and the resulting brown slurry was filtered and rinsed with water (15 ml). The filtrate was filtered again to remove additional solids. The final filtrate was extracted twice with a 1:1 mixture of toluene and heptane (30 ml). The aqueous layer was concentrated in vacuo and resuspended in water (6 ml). The resulting solid was filtered off and the filtrate was subjected to preparative HPLC to give Compound 1 diammonium salt (also known as Compound 1a) (39 mg, 0.050 mmol, 70% yield) as a white solid.

Compound 1a (SpRp, trans) ¹ H NMR (400MHz, CD ₃ OD) δ=9.05(s, 1H), 8.33(s, 1H), 8.25(s, 1H), 8.12(s, 1H), 6.34( br s, 2H), 5.88 (br s, 2H), 5.66 (br d, J=51.6Hz, 1H), 5.59 (brd, J=52.2Hz, 1H), 5.01 (br s, 2H), 4.68-4.34 (m, 6H), 4.07-3.82 (m, 2H), 3.79-3.55 (m, 2H); ³¹ P NMR (162 MHz, CD ₃ OD) δ=55.48 (s, 1P), 55.16 (s, 1P).

Compound 1a preparative HPLC conditions:

Example 1.1--Alternative synthesis of compound 1a

Alternative synthetic routes to compound 1a are listed in Figure 2A and Figure 2B and Figure 2C and reported below.

Stage 1

Compound 129 (570 g, 1.53 mol, 1 wt, 1 vol, 1 equiv) was dissolved in pyridine (2.85 L, 35.2 mol, 4.89 wt, 5.0 vol, 23 equiv). The mixture was cooled to 2.6 °C and treated with 4,4'-dimethoxytrityl chloride (DMTCl; 543 g, 1.60 mol, 0.953 wt, 1.05 equiv). The mixture was stirred at 0-5°C for 2 hours and then warmed to ambient temperature. The reaction was monitored by LC/MS and complete conversion was confirmed after overnight stirring. The reaction mixture was cooled to below 5°C and quenched by treatment with MeOH (124 ml, 3.05 mol, 0.172 wt, 0.217 vol, 2.0 equiv) for 15 min. The mixture was co-evaporated with toluene (2.00 L, 3.04 wt, 3.51 vol) in vacuo, then diluted with a mixture of EtOAc (2.850 L, 4.5 wt, 5.0 vol) and n-heptane (2.85 L, 3.42 wt, 5.0 vol). The organic layer was washed with saturated _NaHCO3 (9 wt% aqueous solution; 2.0 L, 3.5 vol). Additional EtOAc (2.85 L, 4.5 wt, 5.0 vol) was added to completely dissolve the crude product. After stirring for 5 minutes, the two layers were separated. The organic layer was washed with water (2.0 L, 3.5 wt, 3.5 vol). Solids began to slowly precipitate out of the organic layer. The aqueous layer was separated. Then, the organic layer was concentrated to about 1 vol. The crude product was slurried with a mixture of n-heptane (2.00 L, 2.40 wt, 3.51 vol) and toluene (0.50 L, 0.76 wt, 0.88 vol). After stirring for 15 minutes, the pale yellow solid was collected by vacuum filtration. The filter cake was washed sequentially with: (1) a mixture of n-heptane (0.60L, 0.72wt, 1.05vol) and toluene (0.30L, 0.46wt, 0.53vol), then (2) n-heptane (3.00L) , 3.6wt, 5.26vol). The solid was dried without heating for 30 minutes, then transferred to a tray and dried in a vacuum oven at 50°C overnight to afford compound 130 (996.7 g, 1.47 mol, 1.75 wt, 97% yield) as a pale yellow solid.

¹ H NMR (400MHz, chloroform-d) δ=8.99(s,1H), 8.76(s,1H), 8.21(s,1H), 8.04-8.00(m,2H), 7.64-7.59(m,1H) ,7.57-7.50(m,2H),7.41-7.36(m,2H),7.32-7.15(m,7H),6.83-6.76(m,4H),6.31(dd,J=2.5,17.0Hz,1H) ,5.68(ddd,J=2.3,4.7,52.7Hz,1H),4.88-4.77(m,1H),4.26-4.21(m,1H),3.77(s,6H),3.57(dd,J=3.1, 10.9Hz, 1H), 3.43 (dd, J=4.1, 10.7Hz, 1H), 2.60 (br s, 1H)

Stage 1'

Compound 129 (430 g, 1.15 mol, 1 wt, 1 vol, 1 equiv) and imidazole (118 g, 1.73 mol, 0.274 wt, 1.50 equiv) were dissolved in DMF (1.72 L, 3.78 wt, 4.0 vol) and the resulting mixture was cooled to 5°C. TBS-Cl (191 g, 1.27 mol, 0.444 wt, 1.10 equiv) was added. The mixture was stirred at 0-11 °C for 2 hours and allowed to slowly warm to ambient temperature (progress monitored by LCMS). The reaction was complete 6 hours after the addition of TBS-Cl, but was allowed to stir for an additional 20 hours at ambient temperature. The mixture was cooled to 2°C and treated with methanol (93 ml, 74 g, 2.3 mol, 0.17 wt, 0.22 wt, 2.0 equiv) for 10 minutes. The reaction mixture was diluted with a mixture of MTBE (1.72 L, 1.23 kg, 2.96 wt, 4.0 vol) and EtOAc (1.72 L, 1.55 kg, 3.60 wt, 4.0 vol), then saturated _NH4Cl (28 wt% in water; 2.15 L , 5.0vol) dilution. Solids began to slowly settle out of solution. The mixture was warmed to 24°C and water (1.08L, 1.08kg, 2.5wt, 2.5vol) was added (internal temperature=22°C). More solids began to precipitate out of the mixture. Additional water (1.08L, 1.08kg, 2.5wt, 2.5vol) and MTBE (1.40L, 1.04kg, 2.4wt, 3.3vol) were added to the mixture. The off-white solid was collected by vacuum filtration. The reactor was flushed with water (320 ml, 0.74 vol) followed by MTBE (1.80 L, 1.33 kg, 3.10 wt, 4.19 vol) to transfer any remaining solids to the filter. The filter cake was washed sequentially with the following: (1) water (1.80L, 1.80kg, 4.2wt, 4.2vol), (2) water (1.80L, 1.80kg, 4.2wt, 4.2vol), (3) MTBE ( Mixture of 0.90L, 0.67kg, 1.5wt, 2.1vol) and n-heptane (0.90L, 0.62kg, 1.4wt, 2.1vol), (4) MTBE (0.90L, 0.67kg, 1.5wt, 2.1vol) and n-heptane A mixture of alkanes (0.90 L, 0.62 kg, 1.4 wt, 2.1 vol). The recovered solid was vacuum dried at 40°C for 2 days to give compound 133 (483 g, 0.991 mol, 1.12 wt, 86% yield) as a white solid.

¹ H NMR (400MHz, chloroform-d)δ=8.97(s,1H), 8.82(s,1H), 8.36(s,1H), 8.04-8.00(m,2H), 7.64-7.58(m,1H) ,7.56-7.51(m,2H),6.40(dd,J＝2.3,16.0Hz,1H),5.45(ddd,J＝2.7,4.3,53.1Hz,1H),4.75-4.66(m,1H),4.22 -4.17(m,1H),4.07(dd,J=2.3,11.7Hz,1H),3.91(dd,J=2.7,11.7Hz,1H),2.38(dd,J=2.7,7.0Hz,1H), 0.92(s, 9H), 0.11(s, 3H), 0.11(s, 3H).

Stage 2

Compound 130 (993g, 1.47mol, 1wt, 1vol, 1eq) and imidazole (150g, 2.20mol, 0.151wt, 1.5eq) were dissolved in DMF (3.48L, 3.28kg, 3.3wt, 3.5vol), and the The mixture was cooled to 5°C. TBS-Cl (244 g, 1.62 mol, 0.245 wt, 1.10 equiv) was added. The mixture was stirred at 0-5 °C for 2 hours, allowed to slowly warm to ambient temperature and monitored by LCMS. After 17 hours, additional imidazole (100 g, 1.47 mol, 0.10 wt, 1.0 equiv) and TBS-Cl (111 g, 735 mmol, 0.112 wt, 0.50 equiv) were added and stirring was continued for 2 hours at ambient temperature and at 35°C Stir for 2 hours. The resulting mixture was cooled to 13.6 °C and treated with MeOH (119 ml, 2.94 mol, 2 equiv) for 10 min. A separate reactor was charged with ice (5 kg, 5 wt) and saturated _NH4Cl (28 wt% in water; 5.0 L, 5 vol). The reaction mixture was added to an ice/ _NH4Cl mixture. An off-white solid immediately began to precipitate out of solution. An additional 2 kg of ice (2 kg, 2 wt) and water (3.0 L, 3 vol) were added to the mixture. The reaction flask was rinsed with water (0.50 L, 0.5 vol) and the rinse was added to the mixture. To the mixture was added n-heptane (2.00 L, 2 vol) and stirring was continued for 10 minutes. The off-white solid was collected by vacuum filtration. The filter cake was rinsed with: (1) water (4.0L, 4.0vol), (2) water (4.0L, 4.0vol), (3) n-heptane (4.0L, 4.0vol), (4) n-heptane (4.0L, 4.0vol) Heptane (4.0 L, 4.0 vol). The recovered solid was vacuum dried at 45°C for 4 days to give compound 131 (1.095 kg, 1.39 mol, 1.10 wt, 94% yield) as an off-white solid.

¹ H NMR (400MHz, chloroform-d) δ=9.09(s, 1H), 8.78(s, 1H), 8.28(s, 1H), 8.02(d, J=7.4Hz, 2H), 7.63-7.59(m ,1H),7.55-7.50(m,2H),7.37(d,J=7.1Hz,2H),7.29-7.17(m,7H),6.79(d,J=7.9Hz,4H),6.29(dd, J=2.9, 16.2Hz, 1H), 5.60 (ddd, J=2.7, 3.9, 53.1Hz, 1H), 4.78 (ddd, J=4.7, 6.4, 15.8Hz, 1H), 4.26-4.22 (m, 1H) ,3.77(s,6H),3.58(dd,J=3.1,10.9Hz,1H),3.26(dd,J=3.7,10.7Hz,1H),0.85(s,9H),0.10(s,3H), 0.02(s,3H)

Stage 3

Compound 131 (1000 g, 1.27 mol, 1 wt, 1 vol, 1 equiv) and trans-2-butene-1,4-diol (olefin geometry confirmed by ¹ H-NMR; 335 g, 3.80 mol, 0.335 wt, 3.0 equiv) was azeotroped twice with THF (3.0 L, 3.0 vol). The residue was dissolved in a mixture of THF (10 L, 10 vol) and toluene (15 L, 15 vol). Triphenylphosphine (432 g, 1.65 mol, 0.432 wt, 1.3 equiv) was added and the reaction mixture was cooled to -5°C. DIAD (0.320 L, 1.65 mol, 333 g, 0.333 wt, 0.320 vol, 1.3 equiv) was added slowly over 20 minutes while keeping the internal temperature below 5°C. The reaction was stirred at 0-5 °C for 1 hour and monitored by LCMS. The ice bath was removed and the mixture was allowed to warm to room temperature. After overnight stirring (17 hours), triphenylphosphine (83 g, 0.32 mol, 0.083 wt, 0.25 equiv) and DIAD (62 ml, 0.32 mol, 64 g, 0.064 wt, 0.062 vol, 0.25 equiv) were added. After an additional 1 h at room temperature, the reaction mixture was diluted with MTBE (10 L, 10 vol), washed twice with half-saturated NaCl (18 wt% in water; 2 x 4 L), and concentrated in vacuo to a thick oil. The mixture was redissolved in a mixture of MTBE (4.00 L, 4 vol) and n-heptane (0.50 L, 0.5 vol), then cooled to 0 °C. Triphenylphosphine oxide seeds were added to the solution. Solids began to slowly precipitate out of solution and were stirred overnight. The white solid was collected by vacuum filtration and rinsed with MTBE (2 L, 2 vol) to isolate 540 g of triphenylphosphine oxide. The filtrate was concentrated and passed through Biotage 150L KP-Sil ( _SiO2 5kg; pretreated with 1% TEA in Hep/EtOAc; eluent: Heptane/EtOAc (48L 33% EtOAc plus 1% TEA, 24L 50% EtOAc plus 1% TEA, 24L of 66% EtOAc plus 1% TEA) → 100% EtOAc plus 1% TEA) for purification. The column was monitored by TLC (2:1 EtOAc/n-heptane). The clean product fractions were combined and concentrated in vacuo to give compound 132 as an off-white foam solid (634 g, containing 14 wt% DIAD derived by-product, neat 545 g, 0.63 mol, 50% adjusted yield). The mixture fractions were combined and concentrated in vacuo to give a pale yellow foamy solid (750 g) which was passed through Biotage 150MHP-Sphere (2.5 kg _SiO2 ; pretreated with 1% TEA in Hep/EtOAc; sample loaded with toluene eluent) : Hep/EtOAc/1% TEA (12L 50% EtOAc plus 1% TEA, 16L 66% EtOAc plus 1% TEA)→EtOAc plus 1% TEA) for repurification. The column was monitored by TLC (2/1/0.03 EtOAc/n-hep/TEA). The clean product fractions were combined and concentrated in vacuo to give additional compound 132 as a pale white foam solid (206 g, 0.24 mol, 18% yield).

¹ H NMR (400MHz, chloroform-d) δ=8.58(s, 1H), 8.10(s, 1H), 7.43-7.37(m, 2H), 7.32-7.28(m, 2H), 7.24-7.15(m, 8H), 7.03-6.98(m, 2H), 6.78-6.73(m, 4H), 6.18(dd, J=2.7, 17.2Hz, 1H), 5.88(td, J=5.5, 15.6Hz, 1H), 5.77 (td, J=5.1, 15.6Hz, 1H), 5.60 (ddd, J=2.7, 4.3, 53.1Hz, 1H), 5.03-4.96 (m, 2H), 4.91 (ddd, J=4.5, 6.6, 16.6Hz) ,1H),4.18-4.14(m,1H),3.88-3.82(m,2H),3.78(s,6H),3.52(dd,J=2.7,10.9Hz,1H),3.14(dd,J=3.5 , 10.9Hz, 1H), 0.85(s, 9H), 0.10(s, 3H), 0.01(s, 3H).

Stage 4

Compound 132 (800 g, 0.930 mol, 1 wt, 1 vol, 1 equiv) and compound 133 (522 g, 1.07 mol, 0.652 wt, 1.15 equiv) were azeotropically dried with THF (2×3L, 2×3.8vol) and dried at room temperature was redissolved in THF (9.60 L, 8.45 kg, 12.0 vol) under Triphenylphosphine (317 g, 1.21 mol, 0.396 wt, 1.30 equiv) was added and the mixture was cooled to below -5°C. DIAD (226 ml, 1.16 mol, 235 g, 0.294 wt, 0.283 vol, 1.25 equiv) was added and the internal temperature was below 7°C. The reaction was slowly warmed to room temperature. The reaction was monitored by LCMS. After 21 hours, the reaction mixture was concentrated in vacuo to a thick oil, azeotroped with n-heptane (2.00, 1.37 kg, 1.71 wt, 2.50 vol), then redissolved in MTBE (2.40 L, 1.78 kg, 2.2 wt, 3.0 vol) and n-heptane (800 ml, 547 g, 0.68 wt, 1.0 vol). The solution was seeded with triphenylphosphine oxide and cooled to 5°C, diluted with n-heptane (400ml, 274g, 0.34wt, 0.50vol) and stirred at 5°C for 30 minutes. The white solid precipitate was collected by vacuum filtration and rinsed with a 2:1 (v/v) mixture of MTBE and n-heptane (1.8 L) to give triphenylphosphine oxide (455 g). The filtrate was concentrated in vacuo and passed through Biotage 150L KP-Sil ( _SiO2 5kg; pretreated with 1% TEA; sample loaded by dissolving in toluene eluent: 9:1 heptane/EtOAc (16L) plus 15TEA, 3.6:1 (46L), 2:1 (20L) plus 1% TEA, 1:1 (30L) plus 1% TEA, and 100% EtOAc (16L) plus 1% TEA). The combined clean product fractions were concentrated in vacuo to give compound 134 (662.2 g) as an off-white solid foam. The mixture fractions were combined and concentrated in vacuo (480 g). The white insoluble solid formed by dilution with toluene (300 ml) prior to loading into the Biotage 150L was removed by vacuum filtration. Toluene soluble material was passed through Biotage 150M HP-Sphere ( _SiO2 2.5kg (pre-treated with 1% TEA); samples were loaded with toluene; eluent: 2:1 heptane/EtOAc (26L) w/1% TEA, 1:1 (25L) w/1% TEA, 1:4 (34L) w/1% TEA) for purification. The column was monitored by TLC (1:1 heptane/EtOAc). The combined clean product fractions were concentrated in vacuo to give additional compound 134 as an off-white solid foam (165.5 g, total 662.2 + 165.5 g = 827.7 g, 930 mmol, 1.03 wt, 67% yield).

¹ H NMR (400MHz, chloroform-d) δ=8.47(s, 1H), 8.39(s, 1H), 8.20(s, 1H), 8.01(s, 1H), 7.38-7.31(m, 5H), 7.27 -7.19(m,6H),7.14-7.06(m,3H),6.93-6.87(m,2H),6.76(d,J=8.6Hz,4H),6.26(dd,J=2.0,16.0Hz,1H ),6.15(dd,J＝2.7,17.2Hz,1H),5.86(dd,J＝4.7,15.2Hz,1H),5.80(dd,J＝4.7,15.2Hz,1H),5.51(ddd,J＝ 2.7,4.3,52.8Hz,1H),5.31(ddd,J=2.0,4.3,52.8Hz,1H),4.87(d,J=4.7Hz,2H),4.85-4.81(m,1H),4.79(d , J=4.3Hz, 2H), 4.71-4.59 (m, 1H), 4.20-4.13 (m, 2H), 4.06 (dd, J=2.7, 11.3Hz, 1H), 3.90 (dd, J=2.7, 11.7 Hz, 1H), 3.77(s, 6H), 3.52(dd, J=3.1, 10.9Hz, 1H), 3.18(dd, J=3.9, 10.9Hz, 1H), 0.92(s, 9H), 0.84(s ,9H),0.10(s,3H),0.09(s,6H),0.07(s,3H)

Stages 5-6

To a solution of compound 134 (410.7 g, 309 mmol, 1 wt, 1 vol, 1 equiv) in pyridine (1.23 L, 1.21 kg, 15.2 mol, 2.9 wt, 3.0 vol, 49 equiv) was added diphenyl phosphite (90 ml, 109g, 0.46mol, 0.26wt, 0.22vol, 1.5 equiv). The reaction was stirred at room temperature and monitored by LCMS. After 2 hours (80% conversion), additional diphenyl phosphite (29.9 ml, 36.2 g, 155 mmol, 0.088 wt, 0.073 vol, 0.50 equiv) was added. After an additional hour, additional diphenyl phosphite (6.0 ml, 7.2 g, 31 mmol, 0.018 wt, 0.015 vol, 0.10 equiv) was added and the reaction continued for an additional 0.5 hours (98% conversion). The reaction mixture was added to a mixture of saturated _NaHCO3 (9 wt% in water; 2.1 L, 5 vol) and water (1.0 L ml, 2.5 vol) while maintaining the internal temperature at 4.7-12 °C. The reactor was flushed with a small volume of EtOAc. Stirring was continued for 30 minutes at room temperature and the reaction was monitored by LCMS (100% conversion). The reaction mixture was extracted twice with a 1:1 mixture of EtOAc and MTBE (2 x 8.2 L, 2 x 20 vol). The combined organic layers were washed with water (4.1 L, 10 vol), concentrated in vacuo, and azeotroped with toluene (3 x 4.1 L, 3 x 10 vol; continuous feed) to remove pyridine to give compound 135 (0.55 equiv pyridine remaining).

Stage 6 - Crude compound 135 was dissolved in dichloromethane (3.08 L, 4.07 kg, 9.9 wt, 7.5 vol) at ambient temperature. Water (55.7 ml, 0.136 vol, 10 equiv) was added followed by a solution of dichloroacetic acid (77 ml, 120 g, 0.93 mol, 0.29 wt, 0.19 vol, 3.0 equiv) in DCM (3.08 L, 7.5 vol) while maintaining The internal temperature was below 25°C (turned to an orange solution). After 30 minutes, triethylsilane ( _Et3SiH ; 494 ml, 359 g, 3.09 mol, 0.875 wt, 1.20 vol, 10.0 equiv) was added (internal temperature from 18.2°C to 17°C) and stirring was continued for 20 minutes. Triethylamine (431 ml, 313 g, 3.09 mol, 0.762 wt, 1.05 vol, 10.0 equiv) was added (internal temperature from 17.8°C to 22°C). The mixture was concentrated to 1.55 kg (3.8 wt), redissolved in EtOAc (6.2 L, 5.5 kg, 14 wt, 15 vol), washed sequentially with: (1) water (1.0 L, 2.5 vol) and saturated _NaHCO3 (9 wt% aqueous solution, 0.82 L, 2.0 vol). The crude EtOAc solution was stored at -20°C overnight (0.82 L, 2.0 vol) and the next day, the solution was concentrated in vacuo at 25°C. The crude mixture thus obtained (654 g) was triturated with: (1) n-heptane (3.01 L, 7.5 vol), (2) n-heptane (2.46 L, 6.0 vol) and toluene (0.82 L, 2.0 vol) mixture. A portion of the solution (supernatant) was decanted and the solid remaining on the bottom was dissolved in acetonitrile (4.1 L, 10 vol). The mixture was concentrated in vacuo at 25°C and azeotroped twice with acetonitrile to afford compound 136. The product was used in subsequent stages without purification (assuming 100% theoretical yield).

Stage 7

Stage 7a Compound 136 (337 g, 309 mmol, 1 wt, 1 vol, 1 equiv) was dissolved in dry pyridine (13.5 L, 13.2 kg, 39 wt, 40 vol) at room temperature. Triethylamine (129ml, 927mmol, 94g, 0.28wt, 0.38vol, 3.0 equiv) was added followed by 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphine 2- oxide (DMOCP; 103 g, 556 mmol, 0.31 wt, 1.80 equiv). The resulting mixture was stirred at ambient temperature for 30 minutes and monitored by LCMS (100% conversion) to yield compound 137.

Stage 7b TEA (129ml, 927mmol, 94g, 0.28wt, 0.38vol, 3.0eq), water (100ml, 5.56mol, 0.30wt, 0.30wt, 18eq) and sulfur (34.7g, 1.08mol, 0.10wt, 3.5 equiv.) was added to the above mixture of compound 137. After 90 minutes (100% conversion), _NaHCO3 (9 wt% aqueous solution; 3.37 L, 10 vol) was added while keeping the internal temperature below 30°C (16.6°C-27°C). The resulting mixture was filtered to remove salts. The filtrate mixture was concentrated in vacuo, diluted with MTBE (5.1 L, 15 vol), and washed twice with NaCl (30 wt% in water; 2 x 1.35 L, 2 x 4 vol). The insoluble solids were filtered off and the filtrate was concentrated in vacuo and azeotroped with toluene (4.0 L, 12 vol). The resulting solid was removed by filtration and the crude mixture was dissolved in toluene and passed through Biotage 150L KP-Sil ( _SiO2 5kg; pretreated with Hep/EtOAc/TEA (1.5/1.5/0.03CV); eluted with: EtOAc /TEA (3/0.03CV), EtOAc/MeOH/TEA (4/0.2/0.04CV), EtOAC/MeOH/TEA (2/0.2/0.02CV)). The column was monitored by TLC (EtOAC/MeOH/TEA=9/1/0.1). Fractions containing the Sp isomer were combined and concentrated in vacuo to give compound 138 (Sp isomer; 154 g, 128 mmol, 0.46 wt, 41.3% yield) as a pale pink foamy solid. Fractions containing the Rp isomer were combined and concentrated in vacuo to give compound 240 as a pale pink foam solid (Rp isomer; 64 g, 53 mmol, 0.19 wt, 17% yield).

Compound 138 (Sp isomer):

¹ H NMR (400MHz, chloroform-d) δ=8.51(s, 1H), 8.50(s, 1H), 8.22(s, 1H), 8.14(s, 1H), 7.49-7.44(m, 2H), 7.38 -7.27(m,4H),7.25-7.21(m,2H),7.14(t,J=7.1Hz,2H),6.44(dd,J=2.5,13.9Hz,1H),6.18(d,J=15.2 Hz,1H),5.78(td,J＝6.3,15.6Hz,1H),5.69(td,J＝4.7,15.6Hz,1H),5.56(dd,J＝3.9,50.8Hz,1H),5.20-5.06 (m,1H),4.95-4.79(m,4H),4.69(dd,J=4.3,16.0Hz,1H),4.54-4.38(m,3H),4.35(d,J=5.5Hz,1H), 4.32-4.29(m, 1H), 4.05(dd, J=1.6, 11.7Hz, 1H), 3.91(dd, J=3.1, 11.7Hz, 1H), 3.14-3.06(m, 6H), 1.30(t, J=7.4Hz, 9H), 0.91(s, 9H), 0.90(s, 9H), 0.12(s, 3H), 0.08(s, 3H), 0.06(s, 3H), 0.05(s, 3H)

Compound 240 (Rp isomer):

¹ H NMR (400MHz, chloroform-d) δ=8.54(s, 1H), 8.38(s, 1H), 8.33(s, 1H), 8.01(s, 1H), 7.39-7.09(m, 10H), 6.39 (dd,J＝2.3,14.1Hz,1H),6.13(d,J＝17.2Hz,1H),5.72(d,J＝3.1Hz,2H),5.68(dd,J＝4.3,51.2Hz,1H) ,5.43-5.29(m,1H),5.10-4.96(m,3H),4.90-4.83(m,2H),4.78-4.72(m,1H),4.52(ddd,J=3.9,6.6,17.2Hz, 1H), 4.44-4.35(m, 2H), 4.31-4.26(m, 1H), 4.20-4.12(m, 2H), 3.87(dd, J=3.5, 11.7Hz, 1H), 3.79-3.77(m, 1H), 3.15-3.09(m, 6H), 1.33(t, J=7.4Hz, 9H), 0.94(s, 9H), 0.89(s, 9H), 0.13(s, 3H), 0.12(s, 3H ),0.10(s,3H),0.09(s,3H)

Stage 8

Compound 138 (221g, 183mmol, 1wt, 1vol, 1eq) was dissolved in pyridine (530ml, 6.56mol, 519g, 2.3wt, 2.4vol) and TEA (2.65L, 19.0mol, 1.93kg, 8.7wt, 12vol, 104 equivalents) in the mixture. Triethylamine trihydrofluoride (264 ml, 1.62 mol, 262 g, 1.2 wt, 1.2 vol, 8.9 equiv complex, 27 equiv HF) was added and the mixture was stirred at room temperature while monitoring conversion by LCMS. After 3 hours (97% conversion), methoxytrimethylsilane (TMSOMe; 1.40 L, 10.2 mol, 1.06 kg, 4.8 wt, 6.3 vol, 55 equiv) was added and stirring continued for 30 minutes. A sticky solid coats the reactor. Pour off the solution portion (supernatant). The solid was triturated twice with toluene (2 x 2.2 L, 2 x 10 vol; decanted supernatant). The crude solid remaining in the reactor was dissolved in dichloromethane (2.2 L, 10 vol) and washed with _NH4Cl (28 wt% in water; 2.2 L, 10 vol). The aqueous layer was back extracted with dichloromethane (2.2 L, 10 vol). The combined organic layers were washed with a mixture of NaCl (36 wt% in water; 1.1 L, 5 vol) and water (1.1 L, 5 vol), then concentrated in vacuo to give compound 139 as a brown dry foam (152 g, 155 mmol, 0.70 wt, 85 %Yield). The crude product was taken to the next step without purification.

Stage 9

Compound 139 (150 g, 153 mmol, 1 wt, 1 vol, 1 equiv) was azeotroped with acetonitrile (4 L, 27 vol) and then redissolved in acetonitrile (1.05 L, 0.83 kg, 5.5 wt, 7.0 vol) at room temperature. 2-Nitrobenzyl bromide (44.4 g, 205 mmol, 0.30 wt, 1.34 equiv) was added at room temperature and the reaction was monitored by LCMS. After 23 hours (100% conversion), EtOAc (1.50 L, 10 vol), _NH4Cl (28 wt% in water; 300 ml, 2 vol) and water (300 ml, 2 vol) (pH=6) were added and the resulting mixture was placed in Partially vacuum concentrated to a weight of 1.11 kg at 25°C. EtOAc (2.25 L, 15 vol) was added and the mixture was stirred for 5 minutes. Separate the two layers. The aqueous layer was extracted with ethyl acetate (750 ml, 5 vol). The combined organic layers were washed sequentially with: (1) a mixture of NaCl (36 wt% in water; 300ml, 2vol) and water (300ml, 2vol), and (2) water (600ml, 4vol). The organic layer was then concentrated in vacuo and azeotroped with n-heptane (1.50 L, 10 vol). To the crude solid was added MTBE (0.95 L, 6.3 vol) and the mixture was heated at 40°C. The mixture was diluted with EtOAc (300 ml, 2 vol) and slowly cooled to 0 °C. The dense solid was allowed to settle and the supernatant was pumped through the filter frit. The solid was rinsed twice with MTBE (2 x 300 ml, 2 x 2 vol; each time the supernatant was pumped through a filter frit) and dried in vacuo at 40°C overnight to give compound 140 as a pale yellow solid (156 g) . The filtrate was concentrated in vacuo to give a brown oil (17.8 g) which was purified by Biotage Snap-Ultra 340 g (eluent: 0-5% MeOH in EtOAc) to give additional compound 140 (5.8 g) as a pale yellow solid ). Total 156g + 5.8g = 161.8g (152 mmol net, 95% pure, 99% yield)

¹ H NMR (400MHz, chloroform-d) δ=8.46(s,1H), 8.15(s,1H), 8.10(s,1H), 8.09-8.06(m,1H), 7.89(s,1H), 7.54 -7.51(m, 1H), 7.49-7.45(m, 4H), 7.37-7.28(m, 3H), 7.24-7.19(m, 3H), 7.16-7.11(m, 2H), 6.22(d, J= 16.8Hz, 1H), 6.14 (dd, J=2.7, 17.2Hz, 1H), 5.83-5.61 (m, 3H), 5.60-5.48 (m, 1H), 5.07 (dd, J=3.5, 51.6Hz, 1H) ), 5.06-4.96(m, 1H), 4.79(dd, J=4.9, 15.8Hz, 1H), 4.69(d, J=5.9Hz, 2H), 4.67-4.56(m, 1H), 4.48-4.40( m, 3H), 4.37-4.30(m, 1H), 4.27(d, J=5.9Hz, 2H), 4.19-4.13(m, 1H), 3.93-3.85(m, 1H), 3.85-3.78(m, 1H)

Stages 10-11

In stage 10, compound 140 (95% purity, net 73.2 g, 72.3 mmol, 1 wt, 1 vol, 1 equiv.) and N,N,N',N'-tetraisopropylphosphoramidite 2-cyanoethyl ester ( 25.3 ml, 79.5 mmol, 0.33 wt, 0.35 vol, 1.10 equiv) was azeotroped three times (3 x 2 L) with anhydrous acetonitrile, redissolved in dichloromethane (0.73 L, 10 vol), and cooled to 0-5 °C. Diisopropylammonium tetrazolium (6.19 g, 36.1 mmol, 0.085 wt, 0.50 equiv) was added. The resulting reaction mixture was stirred at 0°C for 10 hours, warmed to 10°C over 2 hours, held at 10°C for 10 hours, and warmed to room temperature over 2 hours. The reaction was monitored by LCMS and TLC (EtOAc plus 0.5% TEA). After 18 hours, dry acetonitrile (0.73 L, 10 vol) was added and the mixture was stored at -20°C for 3 days.

Stage 11a The mixture from stage 10 was warmed to ambient temperature and pyridine trifluoroacetate (previously azeotroped twice with pyridine; 41.9 g, 217 mmol) was added via a dropping funnel in portions (100 mL every 30 minutes over 9 hours) , 0.57 wt, 3.0 equiv) and acetonitrile (5.85 L, 80 vol). The reaction was monitored by LCMS. After 13 hours, a solution of N,N,N',N'-tetraisopropylphosphoramidite 2-cyanoethyl ester (5.8 mL, 18 mmol, 0.25 equiv) in acetonitrile (24 mL) was added over 4 hours. The amount of additional reagent was determined based on the remaining compound 140 (approximately 30% based on LCMS). More diol conversion was observed after 6 hours.

Stage 11b added ((dimethylaminomethylene)amino)-3H-1,2,4-dithiazoline-3-thione (DDTT; 20.8 g, 101 mmol, 0.28 wt, 1.4 equiv) and continued stirring 1 hour. The reaction mixture was partially concentrated to about 800 mL and diluted with MTBE (1.46 L, 20 vol), _NaHCO3 (9 wt% in water; 1.1 L, 15 vol) and water (0.37 L, 5 vol). pH=8. The layers were separated and the aqueous layer was extracted with a mixture of MTBE (1.46 L, 20 vol) and EtOAc (1.10 L, 15 vol). The combined organic layers were washed twice with 30% aqueous NaCl (2 x 0.73 L, 2 x 10 vol), concentrated in vacuo at 35°C, and azeotroped with toluene (1.46 L, 20 vol). LCMS and TLC (EtOAc) showed compound 143 (SpRp, desired): compound 241 (SpSp) = 5:1

Pass through Biotage 150M KP-Sil ( _SiO2 2.5kg; eluent: EtOAc/Hep: 2:1 (4CV), 3:1 (2.5CV), 4:1 (2.5CV), 100% EA (3CV), The crude product was purified in 5-10% MeOH 4 CV in EA to give compound 143 (36 g, 31.5 mmol, 44% yield).

Compound 143 (SpRp): ¹ H NMR (400 MHz, chloroform-d) δ=8.59(s,1H), 8.10(s,1H), 8.03-7.99(m,1H), 7.91(s,1H), 7.56- 7.53(m, 2H), 7.49-7.40(m, 5H), 7.35-7.28(m, 2H), 7.24-7.16(m, 4H), 6.92(s, 1H), 6.29(d, J=14.9Hz, 1H), 6.08(d, J=20.7Hz, 1H), 5.97-5.83(m, 1H), 5.76(td, J=4.7, 15.6Hz, 1H), 5.61-5.51(m, 2H), 5.40(d , J=4.3Hz, 1H), 5.29-5.17 (m, 1H), 4.91 (dd, J=7.4, 14.9Hz, 1H), 4.86-4.75 (m, 3H), 4.63 (dd, J=3.7, 9.2 Hz, 1H), 4.58-4.43 (m, 5H), 4.34-4.19 (m, 4H), 2.79 (td, J=5.9, 16.8Hz, 1H), 2.66 (td, J=6.3, 16.8Hz, 1H) .

Compound 241 (SpSp) ¹ H NMR (400 MHz, chloroform-d) δ=8.11 (s, 1H), 8.03 (d, J=8.2 Hz, 1H), 7.94 (s, 1H), 7.90 (s, 1H), 7.61(s, 1H), 7.56-7.40(m, 7H), 7.33-7.28(m, 2H), 7.23-7.17(m, 4H), 6.22(d, J=17.6Hz, 1H), 6.15(d, J=18.8Hz, 1H), 5.85(dd, J=3.5, 51.2Hz, 1H), 5.75-5.45(m, 5H), 4.95-4.23(m, 14H), 2.82(t, J=6.1Hz, 2H) ).

Stage 12

Compound 143 (71.6 g, 62.6 mmol, 1 wt, 1 vol, 1 equiv) was dissolved in 1,4-dioxane (0.43 L, 6 vol). Thiophenol (215ml, 2.09mol, 230g, 3.2wt, 3vol, >30 equiv) was added followed by triethylamine (215ml, 1.54mol, 156g, 2.2wt, 3vol). Some exotherm was observed (internal temperature increased by about 7°C), therefore, a water/ice bath was used for cooling and the internal temperature was controlled below 27°C. The reaction was monitored by LCMS. After 2 hours, MeOH (0.57 L, 8 vol) and _NH4OH (28 wt%; 15 mol, 0.57 L, 8 vol, >200 equiv) were added. The resulting mixture was heated at 50°C for 5 hours, cooled to room temperature, and stirred overnight. After 14 hours, water (0.72 L, 10 vol) was added (no solid observed) and the mixture was extracted three times with a 1:1 (v/v) mixture of n-heptane and toluene (3 x 0.86 L, 3 x 12 vol) , and then extracted with toluene (0.57L, 8vol). The aqueous layer was concentrated in vacuo at 40-50 °C and diluted with water (1.07 L, 15 vol). The resulting slurry was kept at room temperature overnight. The resulting solid was filtered off and rinsed with water (0.36 L, 5 vol). The filtrate, still cloudy, was filtered through celite and a Kuno filter. The cloudiness is still there. HCl (1.0 M in water; 132 ml, 132 mmol, 2.1 equiv) was added over 1 hour and the pH checked (pH<2). Stirring was continued for 1 hour at room temperature, and the mixture was filtered. The filter cake was rinsed with water (8 x 0.20 L), dried in a vacuum oven at 35°C for 2 days, and dried without heating for 1 day to give compound 1 (44.88 g, 60.1 mmol, 0.63 wt, 96 g) as a light orange solid %Yield).

Stage 13

To the free acid compound 1 (22.42 g, 30.03 mmol, 1 wt, 1 vol, 1 equiv) was added ammonia (2.0 M in MeOH; 220 ml, 440 mmol, 10 vol, 15 equiv). EtOH (55 ml, 2.5 vol) was added and the resulting solution was filtered through a Kuno filter (0.45 micron; PTFE) rinsing with a 1:1 (v/v) mixture of MeOH and EtOH (90 mL, 4 vol). The filtrate was concentrated in vacuo at 30°C to give an off-white solid, which was dried overnight at room temperature, triturated with a spatula (easy to break), and further dried in vacuo at room temperature. Then, the separated solid was suspended in toluene (250 ml) and stirred at room temperature for 30 minutes. The solid was then collected by vacuum filtration and rinsed twice with toluene (2 x 50 ml). The solid was then vacuum dried in a vacuum oven to yield 22.4 g of compound 1a (diammonium salt of compound 1).

Recrystallization: Compound 1a (22.14 g, 28.36 mmol, 1 wt, 1 vol, 1 equiv) was dissolved in a mixture of water (664 ml, 30 vol) and ammonium hydroxide (28 wt%; 2.5 ml, 18 mmol, 0.63 equiv) (pH) =9-10) and extracted three times with toluene (3x300ml, 3x14vol), three times with EtOAc (3x200ml, 3x9vol) and three times with toluene (3x300ml, 3x14vol). The resulting aqueous layer was treated with HCl (1.0 M in water; 90 ml, 90 mmol, 3.2 equiv) for 3.5 hours (pH≤2). The mixture was stirred for 30 minutes, then the solid precipitate was collected by vacuum filtration. The filter cake was washed three times with water (3 x 200 ml, 3 x 9 vol) and dried in vacuo overnight. Ammonia (2.0M in MeOH; 250ml, 500mmol, 17.6eq) and ethanol (100ml) were added to the solid and the resulting mixture was concentrated in vacuo until crystals appeared (ca. 100ml) at which point the concentration was stopped and the mixture was stirred for 20 minute. Ethanol (45 mL) was added and the mixture was partially concentrated (45 mL removed). The same operation was repeated two more times, and then the mixture was cooled to 0°C and stirred for 3.5 hours. The white solid was collected by vacuum filtration, washed with cold ethanol (20 mL), then ethyl acetate (2 x 50 mL). The white solid was dried under vacuum at room temperature for 3 days to give compound 1a (16.6 g, 21.3 mmol, 0.75 wt, 75% yield) as a white solid. The filtrate was concentrated in vacuo and dried in vacuo at room temperature for 3 days to give compound 1a (4.16 g, 5.3 mmol, 18% yield) as an off-white solid.

Example 1.2 - ¹ H NMR analysis of compound 1

The ¹ H NMR spectrum of compound 1a is shown in FIG. 3 . The resulting spectrum is:

¹ H-NMR spectrum (400MHz, DMSO-d ₆ , δ _H 2.49ppm, 80°C)

δ(ppm): 3.05-3.13(4H,m),3.70(1H,dd,J=13,5Hz),3.78(1H,dd,J=12,4Hz),4.21-4.24(2H,m),4.28 (1H,m), 4.38(1H,m), 4.53-4.68(2H,m), 5.22(1H,m), 5.76(2H,s), 5.78(1H,m), 6.26(1H,m), 6.29(1H,m), 8.13(1H,s), 8.14(1H,s), 8.36(1H,brs), 8.59(1H,brs).

Example 1.3 - X-ray analysis of compound 1

About 2 mg of Compound 1 was dissolved in 600 uL of water. 120 uL of this solution was placed in another glass vial, which was then stored in a stationary container with 3 mL of MeCN at room temperature for 1 week. This is the _H2O /MeCN vapor diffusion method for sample preparation.

Colorless bulk single crystals (0.1 x 0.1 x 0.1 mm) found in the crystallization solution were dispersed in liquid Parabar 10312 and mounted on a Dual-Thickness MicroMounts ^™ (MiTeGen). Diffraction data were collected on an XtaLAB PRO P200MM007HF (Rigaku) at -160°C using the ω-axis oscillation method using multilayer mirror monochromatic Cu-Kα radiation.

Figure 4A shows the ORTEP map of a compound 1 molecule in the asymmetric unit along with a number of disordered water molecules. Figure 4B shows the crystal structure of one Compound 1 molecule from Figure 4A. Figure 4C shows the crystal structure of another compound 1 molecule shown in Figure 4A.

The crystal structure of compound 1 was solved with a final R factor of 0.1354. The Flack parameter is close to zero (0.083(17)), indicating that the absolute configuration of compound 1 is (R,S). Crystal structure analysis also revealed the presence of many water molecules in the macrochannels of compound 1, suggesting that water molecules can easily slip out of the channels. The analysis also confirmed that the conformations of the two crystallographically independent molecules in the asymmetric unit are nearly identical.

Other parameters of X-ray analysis are shown below:

Example 2 - Synthesis of Compound 2

Compound 106 (the RpRp isomer of Compound 105) obtained from Example 1, Step E, was treated separately by Example 1, Step F and Example 1, Step G to give Compound 2a (the RR isomer of Compound 1a):

Compound 108 (RpRp) ¹ H NMR (400 MHz, CDCl ₃ ) δ=8.12 (dd, J=8.2, 0.8 Hz, 2H), 7.99 (s, 2H), 7.96 (s, 2H), 7.65-7.48 (m, 10H), 7.38-7.33(m, 2H), 7.26-7.24(m, 4H), 6.22(d, J=17.6Hz, 2H), 5.95-5.84(m, 2H), 5.71(dd, J=50.8, 3.9Hz, 2H) 5.73-5.71(m, 2H), 4.88-4.77(m, 4H), 4.59-4.38(m, 8H), 4.19(m, 2H).

Compound 2a (RpRp, trans) ¹ H NMR (400MHz, CD ₃ OD) δ=8.70(s, 1H), 8.49(s, 1H), 8.23(s, 1H), 8.09(s, 1H), 6.34( br s,2H),5.83(br s,2H),5.73-5.53(m,2H),5.38-5.01(m,2H),4.76-4.32(m,6H),3.95(br s,2H),3.69 -3.64 (m, 2H); ³¹ P NMR (162 MHz, CD ₃ OD) δ = 55.57 (s, 1P), 55.32 (s, 1P).

Example 2.1 - X-ray analysis of compound 2

Compound 2 (0.5 mg) was weighed and dissolved in acetonitrile/28% ammonia solution. The solution was then stored at room temperature with the lid loosely secured. Rod-like crystals appeared after 2 weeks.

Colorless bulk single crystals (0.1 x 0.1 x 0.1 mm) found in the crystallization solution were dispersed in liquid Parabar 10312 and mounted on a Dual-Thickness MicroMounts ^™ (MiTeGen). Diffraction data were collected on an XtaLAB PRO P200MM007HF (Rigaku) at -160°C using the ω-axis oscillation method using multilayer mirror monochromatic Cu-Kα radiation.

Figure 4D shows the ORTEP plot of the compound 2 molecule.

Other parameters of this X-ray analysis are shown below:

Example 3 - Synthesis of Compound 3

Compound 109 (SpSp isomer of Compound 105) obtained from Example 1, Step E was treated separately by Example 1, Step F and Example 1, Step G to give Compound 3a (the SS isomer of Compound 1a):

Compound 109 (SpSp isomer of compound 105): ¹ H NMR (400MHz, chloroform-d) δ=8.52(s, 2H), 7.99(d, J=8.2Hz, 2H), 7.91(s, 2H), 7.63-7.40(m, 10H), 7.35-7.28(m, 2H), 7.23-7.16(m, 4H), 6.10-5.93(m, 4H), 5.92-5.75(m, 2H), 5.62-5.50(m ,2H),5.26-5.16(m,2H),5.09-5.03(m,2H),4.98-4.91(m,4H),4.61-4.25(m,10H)

Compound 3a (SpSp, trans): ¹ H NMR (400 MHz, CD ₃ OD) δ=8.97 (br s, 1H), 8.84 (br s, 1H), 8.21 (br s, 2H), 6.31 (br s, 2H), 6.08(br d, J=53.5Hz, 1H), 5.89(br s, 2H), 5.63(brd, J=52.4Hz, 1H), 5.13-4.96(m, 2H), 4.72-4.32(m , 6H), 4.01 (br d, J=9.8Hz, 2H), 3.67 (br s, 2H).

Example 4--Synthesis of compound 4a

Compound 107 obtained from Example 2, Step F was treated separately by Example 1, Step G to give Compound 4a, which was isolated by silica gel chromatography as only the cis isomer.

Compound 107: ¹ H NMR (400 MHz, chloroform-d) δ=8.13 (s, 2H), 8.12-8.08 (m, 2H), 7.91 (s, 2H), 7.65-7.55 (m, 4H), 7.54-7.45 (m,6H),7.33-7.27(m,2H),7.23-7.16(m,4H),6.14(d,J=17.6Hz,2H),5.88(dd,J=3.9,50.8Hz,2H), 5.76-5.61(m, 4H), 5.21-4.99(m, 4H), 4.60-4.46(m, 4H), 4.45-4.37(m, 2H), 4.30-4.13(m, 4H), 3.49(d, J =5.1Hz, 2H)

Compound 4a (RpRp, cis 1a, Compound 4a): ¹ H NMR (400 MHz, methanol-d ₄ ) δ = 8.48 (br s, 2H), 8.02 (br s, 2H), 6.29 (d, J = 14.8 Hz ,2H),5.99(br s,2H),5.43(d,J＝51.2Hz,2H),5.03-4.88(m,2H),4.43(br d,J＝11.7Hz,2H),4.32(br d , J=9.4Hz, 2H), 4.27-4.17 (m, 2H), 4.21-4.02 (m, 2H), 3.97 (brdd, J=6.1, 12.3Hz, 2H).

Example 5--Synthesis of Compound 5

The cis isomer obtained from Example 1, Step F was treated separately by Example 1, Step G to give compound 5a.

Compound 111 (cis isomer of compound 107): ¹ H NMR (400 MHz, chloroform-d) δ=8.48 (s, 1H), 8.15-8.09 (m, 1H), 8.03 (d, J=8.2 Hz, 1H), 7.94(s, 1H), 7.75(s, 1H), 7.65-7.06(m, 11H), 6.07(d, J=17.2Hz, 1H), 5.98(d, J=20.3Hz, 1H), 5.97-5.79(m, 1H), 5.84(dd, J=3.5, 51.2Hz, 1H), 5.54-5.47(m, 1H), 5.50(dd, J=3.9, 52.0Hz, 1H), 5.38-5.21( m,2H),5.18-5.02(m,2H),5.02-4.95(m,1H),4.78-4.69(m,1H),4.60-4.16(m,10H)

Compound 5a (SpRp, 1acis): ¹ H NMR (400 MHz, methanol-d ₄ ) δ=8.88(brs,1H), 8.51(brs,1H), 8.16(s,1H), 8.05(s,1H) ),6.38(d,J＝15.6Hz,1H),6.33(d,J＝14.1Hz,1H),6.14-6.09(m,2H),6.01(d,J＝49.2Hz,1H),5.42(d , J＝49.6Hz, 1H), 5.02-4.87(m, 2H), 4.76-3.92(m, 8H), 3.75-3.56(m, 2H)

Example 6--Synthesis of compound 6a (SpRp)

To a solution of compound 1a (1.5 mg, 2.0 μmol) in MeOH/H ₂ O (0.6/0.5 ml) was added palladium hydroxide on carbon (20 wt% dry basis, 2 mg) at ambient temperature. The resulting mixture was treated with hydrogen (balloon) while monitoring the reaction by LCMS. After complete consumption of starting material, the mixture was filtered and rinsed with methanol until all product was removed. The filtrate was concentrated in vacuo and the residue was dissolved in water (1 ml). RHPLC purification afforded compound 6a (0.9 mg).

LCMS (MS m/z 749.2[M+H] ⁺ )

Example 7--Synthesis of Compound 9

To a solution of 107 (3.7 mg, 3.02 μmol) in EtOH (1.5 mL) was added ammonium hydroxide (1 mL). The resulting mixture was heated at 50°C for 8 hours and cooled to ambient temperature. The solvent was removed under reduced pressure. The residue was treated with 2 mL of water, and the resulting solid was filtered off. The filtrate was subjected to preparative HPLC to give compound 9a (2.5 mg).

Compound 9a: ¹ H NMR (400 MHz, methanol-d ₄ ) δ = 8.67 (br s, 1H), 8.50 (br s, 1H), 8.25 (br s, 1H), 8.12 (br s, 1H), 6.47-6.28 (m,2H),5.92-5.79(m,2H),5.74(d,J=50.8Hz,1H),5.32(d,J=52.4Hz,1H),5.16-4.95(m,2H),4.71- 4.25 (m, 6H), 4.17-3.97 (m, 2H), 3.83-3.61 (m, 2H).

LCMS: MS m/z 715.2 [M+H] ⁺ .

Example 8--Synthetic route of compounds 8a, 11a and 12a

Compounds 8, 11 and 12 were prepared by the same reaction sequence as described in Example 1 using compound 112 and compound 102 as starting materials.

Preparative HPLC Conditions for Fractions A and B

Compound 8a: Fraction A Example 1, Step G was continued to yield two isomers, which were separated by HPLC. Compound 8a was the faster running isomer (retention time: 4.1 minutes), while compound 37a (retention time: 4.5 minutes) was the slower running isomer.

¹ H NMR (400MHz, methanol-d ₄ )δ=9.01(s, 1H), 8.61(s, 1H), 8.22(s, 1H), 8.20(s, 1H), 6.34(d, J=10.2Hz, 1H), 6.31(d, J＝10.9Hz, 1H), 5.92(dd, J＝2.7, 51.2Hz, 1H), 5.85-5.71(m, 2H), 5.37(d, J＝51.6Hz, 1H), 4.81-4.56(m,6H),4.52(br d,J=12.1Hz,1H),4.42(brd,J=9.4Hz,3H),4.05(dd,J=4.1,11.9Hz,1H),3.95( br dd,J=4.3,12.1Hz,1H),3.94-3.84(m,1H),3.62(br dd,J=4.9,15.4Hz,1H),2.71-2.58(m,1H),2.46-2.33( m,1H)

³¹ P NMR (162 MHz, methanol-d ₄ ) δ=55.36(s, 1P), 55.18(s, 1P)

LCMS: MS m/z 761.2 [M+H] ⁺

Compound 37a: ¹ H NMR (400 MHz, methanol-d ₄ ) δ = 8.95(s, 1H), 8.40(s, 1H), 8.18(s, 1H), 8.15(br s, 1H), 6.34(d, J ＝5.5Hz,1H),6.31(d,J＝6.6Hz,1H),5.96-5.68(m,3H),5.40(dd,J＝2.7,51.6Hz,1H),4.85(s,3H),4.58 (br d, J=12.1Hz, 1H), 4.49 (br d, J=12.1Hz, 1H), 4.43-4.33 (m, 3H), 4.29 (dd, J=8.6, 14.1Hz, 1H), 4.05 ( dd,J=4.5,11.9Hz,1H),3.95(dd,J=4.9,12.3Hz,1H),3.94-3.86(m,1H),2.67-2.55(m,1H),2.52-2.40(m, 1H).

Preparative HPLC conditions for compounds 8a/37a:

Fraction B continued Example 1, Step G, yielding two isomers, which were separated by preparative HPLC as described below. Compound 11a was the faster running isomer (retention time: 11.2 minutes), while compound 12a was the slower running isomer (retention time: 12.1 minutes).

Compound 11a: ¹ H NMR (400 MHz, methanol-d ₄ ) δ=8.63 (br s, 2H), 8.18 (br s, 1H), 8.17 (brs, 1H), 6.34 (d, J=13.3 Hz, 1H) ,6.32(d,J＝12.9Hz,1H),5.86-5.65(m,2H),5.48(d,J＝48.5Hz,1H),5.35(d,J＝43.8Hz,1H),4.84-4.53( m,6H),4.47-4.36(m,2H),4.05-3.91(m,2H),3.96-3.85(m,1H),3.70-3.54(m,1H),2.66-2.54(m,1H), 2.43-2.30(m,1H)

Compound 12a: ¹ H NMR (400 MHz, methanol-d ₄ ) δ = 8.56 (br s, 1H), 8.41 (s, 1H), 8.17 (s, 1H), 7.95 (br s, 1H), 6.35 (d, J＝14.8Hz, 1H), 6.31(d, J＝15.2Hz, 1H), 5.82-5.65(m, 2H), 5.50(d, J＝51.2Hz, 1H), 5.36(d, J＝53.9Hz, 1H), 4.74-4.33 (m, 8H), 4.26-4.16 (m, 1H), 4.07-3.93 (m, 3H), 2.64-2.44 (m, 2H).

Preparative HPLC conditions for compounds 11a/12a:

Example 9--Synthetic route of compounds 13 and 14

Using compound 114 and compound 115 as starting materials, compounds 13 and 14 were prepared by the same reaction sequence as described in Example 1 plus a TBS deprotection step.

Compound 116: ¹ H NMR (400 MHz, chloroform-d) δ=9.36(s,1H), 9.18(s,1H), 8.77(s,1H), 8.76(s,2H), 8.07(br dd, J= 1.8,7.6Hz,3H),8.05-8.00(m,2H),7.99(s,1H),7.87-7.82(m,1H),7.70-7.65(m,1H),7.63-7.55(m,3H) ,7.55-7.41(m,6H),7.39-7.35(m,1H),7.30-7.26(m,1H),6.37(d,J=8.6Hz,1H),5.89(d,J=4.7Hz,1H ),5.36-5.27(m,2H),5.15(ddd,J=3.7,8.4,11.7Hz,1H),4.64-4.43(m,4H),4.43-4.38(m,1H),4.37-4.30(m ,1H),4.23-4.13(m,2H),4.11-3.98(m,2H),3.97-3.86(m,1H),0.97(s,9H),0.83(s,9H),0.25(s,3H ), 0.18(s, 3H), 0.16(s, 3H), -0.13(s, 3H).

Compound 13a: ¹ H NMR (400 MHz, methanol-d ₄ ) δ = 8.78 (br s, 1H), 8.47 (brs, 1H), 8.21 (brs, 1H), 8.08 (br s, 1H), 6.44-6.21 ( m,1H),6.19-6.03(m,1H),6.01-5.78(m,2H),5.38-4.95(m,2H),4.67-4.45(m,5H),4.45-4.35(m,1H), 4.34-4.29 (m, 1H), 4.25 (br d, J=11.3 Hz, 1H), 4.18-3.89 (m, 2H), 3.88-3.54 (m, 2H).

³¹ P NMR (162MHz, methanol-d ₄ )δ=56.89(br s,1P),56.37(br s,1P)

Compound 117 (Rp ₁ Rp ₂ isomer of compound 116): ¹ H NMR (400 MHz, chloroform-d) δ=9.36(br s, 1H), 9.11(br s, 1H), 8.98(s, 1H), 8.87(s, 1H), 8.78(s, 1H), 8.14-7.96(m, 6H), 7.72-7.27(m, 13H), 6.44(d, J=8.6Hz, 1H), 5.84(d, J= 6.6Hz,1H),5.59-5.50(m,1H),5.48-5.44(m,1H),5.33-5.24(m,1H),4.75-3.85(m,11H),0.90(s,9H),0.79 (s,9H),0.20(s,3H),0.11(s,3H),0.07(s,3H),-0.21(s,3H)

Compound 14a (Rp ₁ Rp ₂ isomer of compound 13a)

LCMS: MS m/z 743.17[M+H] ⁺

Example 10--Synthetic route of compounds 15 and 16

To a solution of compound A (2.63 g, 2.46 mmol) in MeCN (26.3 ml) was added tert-butylamine (13.15 ml, 124.1 mmol). After stirring at ambient temperature for 1 hour, the reaction mixture was concentrated in vacuo and azeotroped with MeCN. The residue was dissolved in MeCN (52.6 ml) and treated with 1-(bromomethyl)-2-nitrobenzene (1.064 g, 4.925 mmol) and triethylamine (0.755 ml, 5.42 mmol). After complete reaction (monitored by LC/MS), the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography (66% ethyl acetate/n-heptane to 100% ethyl acetate) to give 0.226 g of compound 118. The isolated product was dissolved in DMF (5 mL) and allyl bromide (0.046 ml, 0.528 mmol) and potassium carbonate (0.073 g, 0.528 mmol) were added. The resulting mixture was stirred at ambient temperature while monitoring progress by LCMS. After 19 hours, a 1/1 mixture of MTBE/EtOAc (12/12 mL), saturated aqueous _NH4Cl (15 mL) and water (10 mL) were added. The organic layer was separated, washed twice with brine (5 mL), dried over _MgSO4 , and concentrated in vacuo. The residue was purified by silica gel column chromatography (50% ethyl acetate/n-heptane to 100% ethyl acetate) to give 37 mg of bisallylated product. The isolated product (37 mg, 0.027 mmol) was dissolved in toluene (55 mL) and heated to gentle reflux (120-125 °C oil bath). Add the second generation Hoveyda-Grubbs catalyst ((1,3-bis-(2,4,6-trimethylphenyl)-2-imidazolidinylidene)dichloro(o-isopropoxyphenylmethylene) ) ruthenium; can Obtained as Cat. No. 569755; CAS 301224-40-8; 8.5 mg, 0.014 mmol) and a solution of quinone (5.9 mg, 0.054 mmol) in toluene (10 mL). The mixture was heated to reflux and the progress of the reaction was monitored by LC/MS. After complete consumption of starting material, the mixture was cooled to ambient temperature and treated with DMSO (0.059 ml, 0.81 mmol) for 15 hours. The resulting mixture was concentrated in vacuo and purified by silica gel column chromatography ( _SiO2 10 g, 66% ethyl acetate in n-heptane to 100% ethyl acetate) to give compound 119 (2.2 mg).

Compound 119: ¹ H NMR (400 MHz, chloroform-d) δ=8.49 (s, 1H), 8.12 (d, J=7.8 Hz, 1H), 8.05-8.02 (m, 1H), 8.02 (s, 1H), 7.82(s, 1H), 7.70-7.10(m, 17H), 6.27(d, J=16.8Hz, 1H), 6.15-6.05(m, 1H), 5.97(d, J=8.2Hz, 1H), 5.94 -5.72(m,3H),4.94(br s,2H),4.86-4.69(m,2H),4.67-4.60(m,1H),4.60-4.44(m,4H),4.39-4.33(m,1H) ),4.31-4.10(m,6H),0.96(s,9H),0.25(s,3H),0.19(s,3H)

Compound 119 continued with Step G in Example 1, followed by TBS deprotection with TEA 3HF to afford compound 15a.

Compound 15a: ¹ H NMR (400 MHz, methanol-d ₄ ) δ = 8.76 (br s, 1H), 8.44 (brs, 1H), 8.24 (brs, 1H), 8.07 (br s, 1H), 6.45-6.19 ( m,2H),6.12-5.75(m,2H),5.54(br d,J=51.6Hz,1H),5.67-5.06(m,2H),4.66-4.37(m,3H),4.47-4.37(m ,1H),4.36-4.22(m,2H),4.17-3.94(m,2H),3.94-3.80(m,1H)3.80-3.59(m,2H)

Compound 16 was obtained from compound B (RR isomer of compound A) by the same sequence as described in compound 15.

Compound 120 (Rp ₁ Rp ₂ isomer of compound 119): ¹ H NMR (400 MHz, chloroform-d) δ = 8.89 (s, 1H), 8.10 (d, J = 8.2 Hz, 1H), 8.01 (d, J=8.2Hz, 1H), 7.85-7.74(m, 4H), 7.69-7.61(m, 2H), 7.58(s, 1H), 7.55-7.48(m, 1H), 7.45-7.30(m, 6H) ,7.22-7.15(m,1H),7.13-7.04(m,3H),6.76-6.68(m,1H),6.31-6.22(m,1H),6.22(d,J=16.0Hz,1H),6.12 -5.99(m,2H),5.94(d,J=8.2Hz,1H),5.92-5.81(m,1H),5.67(dd,J=3.5,51.2Hz,1H),5.08-4.93(m,2H) ),4.87-4.77(m,1H),4.60-4.22(m,8H),4.21-4.16(m,1H),4.11-4.02(m,1H),3.94(br d,J=11.7Hz,1H) ,3.66(dd,J＝4.7,11.3Hz,1H),0.90(s,9H),0.09(s,3H),0.08(s,3H)

Compound 16a: ¹ H NMR (400 MHz, methanol-d ₄ ) δ=8.54 (br s, 1H), 8.24 (brs, 1H), 8.17 (brs, 1H), 8.08 (br s, 1H), 6.44-6.23 ( m,2H),6.07-5.68(m,2H),5.43(d,J=50.4Hz,1H),5.31-5.05(m,2H),4.69-4.26(m,6H),4.19-3.90(m, 2H),3.90-3.57(m,3H)

Example 11--Compound Adenine/Guanine (A/G) Analogs-Synthetic Pathway of Compound 21, Compound 22 and Compound 23

Adenine/guanine analogs were prepared by the same reaction sequence as described in Example 1 using compound 121 and compound 101 as starting materials.

Preparative HPLC Conditions for Separation of Compounds C, D, and E

Compound D: ¹ H NMR (400 MHz, chloroform-d) δ=8.60(s, 1H), 8.27(s, 1H), 8.04-8.01(m, 1H), 8.01(s, 1H), 7.99-7.95(m ,1H),7.80(s,1H),7.63-7.51(m,5H),7.46-7.41(m,1H),7.39-7.30(m,3H),7.25-7.20(m,2H),6.96-6.84 (m,1H),6.19-5.99(m,4H),5.67(dd,J=4.3,52.3Hz,1H),5.47(dd,J=1.4,17.4Hz,1H),5.36-5.24(m,4H) ), 5.14-5.09(m, 1H), 5.08(d, J=5.5Hz, 2H), 5.04-4.98(m, 2H), 4.51-4.30(m, 10H), 4.21-4.13(m, 1H), 2.83-2.67(m,1H),1.16(d,J=7.0Hz,3H),1.14(d,J=6.6Hz,3H)

Compound D Continue with steps F and G of Example 1 to give compound 22.

Compound 22: LCMS: MS m/z 763.07 [M+H] ⁺

Compound C: ¹ H NMR (400 MHz, chloroform-d) δ=9.08(s, 1H), 8.60(s, 1H), 8.07-8.02(m, 2H), 8.02(s, 1H), 7.80(s, 1H) ),7.55-7.30(m,9H),7.24-7.19(m,2H),6.58-6.45(m,1H),6.23-5.97(m,4H),5.97-5.86(m,1H),5.63-5.57 (m,1H),5.54-5.47(m,1H),5.48-5.44(m,1H),5.35-5.30(m,1H),5.29-5.22(m,1H),5.18-5.13(m,2H) ,5.11-5.07(m,1H),5.02-4.97(m,2H),4.51-4.22(m,9H),4.14-4.07(m,1H),2.99-2.85(m,1H),1.20(d, J=6.6Hz, 3H), 1.17(d, J=7.0Hz, 3H)

Compound C continues with steps F and G in Example 1 to give compound 21.

Compound 21: LCMS: MS m/z 763.13 [M+H] ⁺

Compound E: ¹ H NMR (400 MHz, chloroform-d) δ=8.99(s,1H), 8.57(s,1H), 8.02-7.94(m,2H), 7.93(s,1H), 7.82(s,1H) ),7.68-7.63(m,1H),7.60-7.55(m,1H),7.54-7.49(m,2H),7.45-7.31(m,5H),7.25-7.21(m,2H),6.22-5.94 (m,6H),5.56(br dd,J=5.1,51.5Hz,1H),5.54-5.46(m,1H),5.34-5.29(m,1H),5.29-5.22(m,2H),5.16- 5.08(m, 3H), 5.02-4.96(m, 2H), 4.62-4.54(m, 1H), 4.52-4.26(m, 9H), 2.93-2.81(m, 1H), 1.21(d, J=2.3 Hz,3H),1.20(d,J＝2.0Hz,3H)

Compound E continued with steps F and G in Example 1 to give compound 23.

Compound 23: LCMS: MS m/z 763.18 [M+H] ⁺

Example 12--Synthesis of Compound 24

Compound 24a was prepared by the same reaction sequence as described in Example 1 using Compound F.

Compound 24: ¹ H NMR (400 MHz, methanol-d ₄ ) δ = 8.95 (br s, 1H), 8.29 (br s, 1H), 8.23 (brs, 1H), 8.08 (br s, 1H), 6.31 (br s, 1H) d, J＝12.9Hz, 2H), 6.23-6.07(m, 1H), 6.04-5.84(m, 2H), 5.71(br d, J＝50.8Hz, 1H), 5.27-4.32(m, 7H), 4.27-4.16(m, 1H), 4.12(dd, J=5.7, 11.9Hz, 1H), 4.07-3.98(m, 1H), 3.78-3.62(m, 2H), 1.40(d, J=7.0Hz, 3H)

Example 13 - Synthesis of Compound 18, Compound 19 and Compound 20

The synthetic route is shown in Figure 3 .

To 9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-ol (100 mg) at 20°C , 0.37 mmol) and Hunig's base (0.129 ml, 0.74 mmol) in DMF (1 ml) was added BOP (327 mg, 0.74 mmol). The mixture was stirred at 20°C overnight. UPLC-MS indicated that the reaction was complete. Solvents were removed on a high vacuum rotary evaporator. The residue was dissolved in DCM and purified using Biotage (24g silica column, EtOAc in heptane = 0-100%, 10vol, 100%, 10vol) to give 105 mg of BOP adduct in 73% yield .

¹ H NMR (400 MHz, methanol-d ₄ ) δppm 3.74-3.84(m,1H)3.92-4.00(m,1H)4.12-4.19(m,1H)4.62-4.74(m,1H)5.37-5.57(m,1H) 1H)6.41-6.52(m,1H)7.47-7.55(m,1H)7.57-7.64(m,2H)8.03-8.14(m,1H)8.36-8.46(m,1H)8.86(s,1H)

To (2R,3R,4R,5R)-5-(6-((1H-benzo[d][1,2,3]triazol-1-yl)oxy)-9H- at 20°C Purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol (135 mg, 0.349 mmol) in allyl alcohol (3 ml) was added cesium carbonate (500 mg, 1.535 mmol). The mixture was stirred at 20°C for 1 hour. UPLC-MS indicated that the reaction was complete. The reaction was treated with saturated _NaHCO3 /brine and extracted with EtOAc/Hept. The organic layer was dried _{over Na2SO4} and filtered. Solvents and volatile organics were removed from the filtrate on a rotary evaporator. The residue was purified using Biotage (12 g silica column, EtOAc in heptane = 0-100%, 10 vol, 100%, 10 vol) to give 100 mg of the desired allyl adduct in 92% yield.

¹ H NMR (400 MHz, methanol-d ₄ ) δppm 8.56(s,1H)8.47(s,1H)6.29-6.41(m,1H)6.06-6.21(m,1H)5.33-5.53(m,2H)5.22- 5.31(m,1H)5.02-5.14(m,2H)4.56-4.71(m,1H)4.10-4.18(m,1H)3.88-3.96(m,1H)3.70-3.79(m,1H)

To diisopropylphosphoramidite (150 mg, 0.164 mmol) and (2R,3R,4R,5R)-5-(6-(allyloxy)-9H-purin-9-yl) at 20°C To a solution of -4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol (76 mg, 0.246 mmol) in acetonitrile (1069 μl, 20.47 mmol) was added molecular sieves (3A, 150 mg). The mixture was stirred at 20°C for 1 hour, then 1H-imidazole-4,5-dicarbonitrile (38.7 mg, 0.328 mmol) was added at room temperature. The mixture was stirred at room temperature for 1 hour. UPLC-MS indicated that the reaction was complete. (E)-N,N-Dimethyl-N'-(3-thione-3H-1,2,4-dithiazol-5-yl)formamidine (37.0 mg, 0.18 mmol) was added. UPLC-MS indicated that vulcanization was complete within 30 minutes. The reaction was treated with saturated _NaHCO3 /brine and extracted with EtOAc/Hept. The organic layer was dried _{over Na2SO4} and filtered. Solvents and volatile organics were removed from the filtrate on a rotary evaporator. The residue was purified using Biotage (25g silica column, EtOAc in heptane = 0-100%, 15vol, 100%, 10vol).

¹ H NMR (500 MHz, methanol-d ₄ , two diastereomers about 1:1) δ ppm 8.62-8.63 (m, 1H) 8.57-8.58 (m, 1H) 8.48-8.50 (m, 1H) 8.44 -8.46(m,1H)8.40-8.41(m,1H)8.38-8.39(m,1H)8.33-8.35(m,1H)8.25-8.27(m,1H)7.22-7.33(m,11H)7.11-7.20 (m,17H)7.05-7.11(m,4H)6.83-6.92(m,6H)6.65-6.77(m,11H)6.28-6.41(m,5H)6.23-6.28(m,2H)6.10-6.22(m ,5H)6.00-6.10(m,4H)5.89-6.00(m,5H)5.52-5.57(m,2H)5.45-5.52(m,3H)5.41-5.45(m,1H)5.24-5.34(m,4H) )5.13-5.17(m,3H)5.07-5.12(m,5H)4.93-4.98(m,4H)4.88-4.93(m,5H)4.73-4.82(m,3H)4.50-4.59(m,2H)4.36 -4.48(m,4H)4.27-4.36(m,5H)4.16-4.27(m,5H)3.83-4.04(m,5H)3.73-3.78(m,13H)3.64-3.73(m,4H)3.52-3.58 (m,1H)3.45-3.51(m,2H)3.35-3.41(m,2H)3.16-3.23(m,2H)3.08-3.15(m,2H)2.84-2.93(m,2H)2.79-2.83(m ,2H)2.70-2.77(m,2H)2.61-2.69(m,3H)

At 20 °C, to O-((2R,3R,4R,5R)-5-(6-(N-allylbenzamido)-9H-purin-9-yl)-2-((bis (4-Methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl)O-(((2R,3R,4R,5R)-5-(6-( Allyloxy)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl)O-(2-cyanoethyl)phosphorothioate (120 mg, 0.104 mmol) To a solution in acetonitrile (4171 μl, 79.852 mmol) was added molecular sieves (3A, 1 wt). The mixture was stirred at 20°C for 1 hour, then tripyrrolidinephosphine (71.6 μl, 0.311 mmol) was added. Then 0.45M tetrazole in ACN (253 μl, 0.114 mmol) was added in 7 portions at 2 minute intervals. TLC (EtOAc/Hept=2:1, RfSM=0.7, RfProd=0.0-0.6) showed incomplete reaction. Add 2X phosphorylation reagent and tetrazole. The reaction mixture was stirred at room temperature for 10 minutes. Neither UPLC-MS nor TLC showed any SM residues. The reaction mixture was transferred to a flask containing acetonitrile (20.8 ml), water (104 μl, 5.776 mmol) and pyridine trifluoroacetate (421 mg, 2.178 mmol). The mixture was stirred for 10 minutes. UPLC-MS indicated that the desired product had been formed. The reaction mixture was mixed with EtOAc and washed with HCl [0.1N]/brine, then saturated _NaHCO3 /brine to prevent DMT deprotection. The aqueous layer was back extracted with EtOAc (1X). The combined organic layers were dried over Na2SO4 and filtered. The filtrate was freed of solvent and volatiles on a rotary evaporator to give the crude product, which was used in the next step without further purification.

The crude hydrogen phosphonate intermediate was dissolved in DCM (2635 μl, 40.946 mmol) and water (29.5 μl, 1.638 mmol), then dichloroacetic acid (135 μl, 1.638 mmol) in DCM (2635 μl, 40.946 mmol) was added at 20°C 1.638 mmol). The mixture was stirred at 20°C for 5 minutes. UPLC-MS indicated that the reaction was complete. The reaction was neutralized with pyridine (510 μl, 6.308 mmol). Remove volatiles on a rotary evaporator followed by a high vacuum rotary evaporator. The residue was azeotroped once more with pyridine before being used for cyclization.

The crude DMT deprotected hydrogen phosphonate (80 mg, 0.087 mmol) was dissolved in pyridine (1831 μl, 22.639 mmol) followed by the addition of 2-chloro-5,5-dimethyl-1,3,2- Dioxaphosphalane 2-oxide (48.2 mg, 0.261 mmol). The mixture was stirred at room temperature for 10 minutes. UPLC showed no SM peak at 2.1 min. The reaction was quenched by the addition of water (47.1 μl, 2.612 mmol), followed by the immediate addition of 3H-benzo[c][1,2]dithiol-3-one (21.97 mg, 0.131 mmol). After 10 minutes, UPLC showed no starting material remaining. The reaction was stirred at room temperature for 1 hour. No changes were found on UPLC-MS. The reaction mixture was diluted with EtOAc and washed with saturated _NaHCO3 and brine. The organic layer was dried _{over Na2SO4} , then filtered. The residue after solvent evaporation was dissolved in MeOH, then _NH3 - _H2O was added. The mixture was stirred for 5 minutes, then all solvent and volatiles were removed on a rotary evaporator and a high vacuum rotary evaporator.

The CDN intermediate was dissolved in acetonitrile (1484 μl, 28.417 mmol) and 1,4-dioxane (496 μl, 5.797 mmol), then TEA (39.6 μl, 0.284 mmol) and 1-(bromomethyl) were added at 20°C )-2-nitrobenzene (49.1 mg, 0.227 mmol). The mixture was stirred at 20°C for 16 hours. UPLC-MS indicated that the reaction was not complete. Neither TLC (EtOAc) nor HPLC showed any o-nitrobenzyl bromide residues. 1-(Bromomethyl)-2-nitrobenzene (49.1 mg, 0.227 mmol) and TEA (39.6 μl, 0.284 mmol) were added. The reaction was stirred for 5 hours. The reaction was treated with saturated _NaHCO3 /brine and extracted with EtOAc/Hept. The organic layer was dried _{over Na2SO4} and filtered. Solvents and volatile organics were removed from the filtrate on a rotary evaporator. The residue was purified using Biotage (12 g silica column, EtOAc in heptane = 0-100%, 15 vol, 100%, 10 vol, 10% MeOH in EtOAc, 6 vol). Fractions containing the desired product were combined. Evaporation of the solvent gave the desired product. The most polar isomer was separated using Prep-TLC (EtOAc) to give the final product compound 18. The other two isomers were separated using prep-TLC (EtOAc/DCM=1:1, 3 runs). The more polar isomer was converted to compound 19 and the less polar isomer was converted to compound 20.

The most polar product from the first p-TLC (EtOAc) was named compound 128-1: ¹ H NMR (500 MHz, chloroform-d) δ ppm 8.62 (s, 1H) 8.55 (s, 1H) 8.00-8.07 (m,2H)7.99(s,1H)7.96(s,1H)7.56-7.64(m,2H)7.48-7.56(m,3H)7.42-7.48(m,1H)7.34-7.42(m,2H)7.29 -7.34(m,1H)7.18-7.25(m,2H)6.09-6.21(m,3H)5.98-6.09(m,2H)5.63-5.75(m,1H)5.52-5.63(m,1H)5.42-5.51 (m,2H)5.32(dd,J＝10.27,0.98Hz,1H)5.27(dd,J＝17.12,1.47Hz,1H)5.13(d,J＝5.87Hz,2H)5.09-5.12(m,1H) 4.95-5.05(m,2H)4.36-4.54(m,7H)4.23-4.31(m,2H)4.15-4.21(m,1H)

The more polar product from the second p-TLC was named compound 128-2 (EtOAc/DCM=1:1, 3X) ¹ H NMR (400 MHz, methanol-d ₄ ) δ ppm 8.75 (s, 1H) 8.59 ( s,1H)8.43(s,1H)8.39(s,1H)8.01-8.07(m,2H)7.38-7.56(m,7H)7.27-7.35(m,1H)7.15-7.26(m,3H)6.45( d, J=19.91Hz, 1H) 6.38 (d, J=19.52Hz, 1H) 5.95-6.26 (m, 4H) 5.83 (dd, J=51.54, 4.69Hz, 1H) 5.73 (dd, J=51.54, 4.69 Hz,1H)5.46-5.57(m,1H)5.28-5.36(m,1H)5.19-5.28(m,1H)5.16(dt,J=5.56,1.51Hz,2H)5.03-5.10(m,1H)4.95 (br d,J＝5.50Hz,2H)4.41-4.62(m,4H)4.28-4.40(m,2H)4.12-4.27(m,4H)

The less polar product from the second p-TLC was named compound 128-3 (EtOAc/DCM=1:1, 3X) ¹ HNMR (400 MHz, chloroform-d) δ ppm 8.53 (s, 1H) 8.36 (s ,1H)7.95-8.02(m,2H)7.95(s,1H)7.89(s,1H)7.53-7.61(m,2H)7.44-7.53(m,4H)7.36-7.44(m,2H)7.25-7.31 (m,1H)7.12-7.20(m,2H)5.91-6.18(m,4H)5.85-5.91(m,1H)5.72-5.79(m,1H)5.61-5.72(m,1H)5.47-5.57(m ,1H)5.40-5.47(m,1H)5.25-5.32(m,1H)5.15-5.23(m,1H)4.99-5.14(m,3H)4.92(d,J=5.47Hz,2H)4.52-4.60( m,2H)4.37-4.52(m,4H)4.35(d,J=3.13Hz,1H)4.30(d,J=5.47Hz,1H)4.25(d,J=2.74Hz,1H)4.20(d,J =4.69Hz, 1H)

To a refluxing solution of 2-nitrobenzyl protected CDN (10 mg, 8.696 μmol) in toluene (20 ml, 187.761 mmol) was added the second generation Hoveyda-Grubbs catalyst ((1,3-bis-(2,4, 6-Trimethylphenyl)-2-imidazolidineidene)dichloro(o-isopropoxyphenylmethylene)ruthenium; can Obtained as Cat. No. 569755; CAS 301224-40-8; 2.73 mg, 4.348 μmol) and a solution of benzoquinone (2.350 mg, 0.022 mmol) in toluene (2 ml). The resulting solution was refluxed for 4 hours (oil bath temperature 120-125°C). TLC (EtOAc, Rfsm=0.8, RfProd=0.3) and UPLC-MS indicated that the reaction was incomplete. 1 ml of Ru catalyst solution was added. The reaction was refluxed for an additional 2 hours. There is still a lot of unreacted SM remaining. An additional 1 ml of Ru catalyst solution was added. The reaction was refluxed for an additional 2 hours. No SM was detected by reverse phase HPLC. The reaction mixture was cooled to room temperature and quenched with DMSO (0.019 ml, 0.261 mmol). Evaporation of the solvent gave the crude product, which was purified on a silica gel column (10 g silica column, eluent: 50-100%, 15vol, 100%, 10vol). Fractions containing the product with the desired MS were combined and rotary evaporated. The residue was repurified on Prep-TLC (EtOAc).

To pTLC purified bridged CDN (2.5 mg, 2.228 μmol) (trans isomer) in 1,4-dioxane (0.5 ml, 5.845 mmol) was added thiophenol (0.25 μmol) at 20 °C ml, 2.428 mmol) and TEA (0.25 ml, 1.794 mmol). The mixture was stirred at room temperature for 3 hours. UPLC indicated that conversion was complete.

Methanol (1.5ml) was added followed by 29% _NH3 in _H2O (1.0ml). The resulting mixture was heated at 50°C for 6 hours and cooled to room temperature.

The suspension of the reaction mixture was filtered and rinsed with water (25 mL). A precipitate formed in the filtrate after washing with water. The filtrate was filtered again to remove some solids.

The resulting filtrate was extracted with a tol/Hep mixture (3X, 1/1, 25 ml each). The aqueous layer was concentrated in vacuo, then dissolved in water (6 mL). The precipitate was filtered again. UPLC indicated that the desired product had formed. The product was purified using reverse phase HPLC using the same method as for other locked CDN based analogs.

For compound 18, LCMS: MS m/z 748.11 [M+H] ⁺ 746.15 [MH] ^-

For compound 19, LCMS: MS m/z 748.06 [M+H] ⁺ 746.17 [MH] ^-

For compound 20, LCMS: MS m/z 748.09 [M+H] ⁺ 746.21 [MH] ^-

Example 13--Synthesis of Compound 26

Compound 151

To a solution of compound 150 (0.70 g, 2.256 mmol) in pyridine (10.5 ml) was added 4,4'-dimethoxytrityl chloride (0.803 g, 2.369 mmol) at 0 °C. The resulting solution was stirred overnight while warming to ambient temperature. Upon completion (monitored by LCMS), saturated _NH4Cl solution (10 ml) and MTBE (20 mL) were added. The layers were separated and the aqueous layer was extracted with MTBE/EtOAc (3/1, 12 mL). The combined organic layers were washed with 30% aqueous NaCl (5 ml), dried over _MgSO4 , filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography ( _SiO2 25g, 50%-70% EtOAc in n-heptane) to give 0.942g of compound 151.

¹ H NMR (400MHz, chloroform-d) δ=8.47(s, 1H), 8.11(s, 1H), 7.43-7.35(m, 2H), 7.30-7.26(m, 4H), 7.25-7.17(m, 3H),6.82-6.75(m,4H),6.28(dd,J=2.3,17.6Hz,1H),6.22-6.10(m,1H),5.63(ddd,J=2.7,4.7,53.1Hz,1H) ,5.47(qd,J=1.4,17.3Hz,1H),5.31(dd,J=1.2,10.6Hz,1H),5.13(t,J=1.4Hz,1H),5.12(t,J=1.2Hz, 1H),4.89-4.76(m,1H),4.21(td,J=3.2,6.8Hz,1H),3.78(s,6H),3.55(dd,J=2.7,10.9Hz,1H),3.44(dd , J=4.3, 10.9Hz, 1H), 2.29-2.21 (m, 1H).

Compound 152

To a solution of compound 151 (0.942 g, 1.538 mmol) in DMF (7.5 mL) at ambient temperature was added 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (0.927 g, 3.075 mmol), 0.45M 1,2,3,4-tetrazole (2.8 mL, 1.2 mmol) and 1-methylimidazole (30 μl, 0.38 mmol). The resulting solution was stirred at ambient temperature for 4 hours. Upon completion (monitored by LCMS), TEA (0.50 ml, 3.6 mmol), DMF (11.3 mL) and water (1.9 mL) were added. The resulting mixture was extracted three times with n-heptane (3 mL each). The DMF layer was diluted with water (4 ml) and extracted with a toluene/n-heptane mixture (1/1, 10 mL). The combined organic layers were washed twice with 30% aqueous NaCl (3 mL each), dried over _MgSO4 , filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography ( _SiO2 50 g, 33%-40% EtOAc in n-heptane plus 1% TEA) to give 1.115 g of compound 152 (3:2 diastereomer) as a white foamy solid structure mixture).

Compound 153

A mixture of compound 152 (1.115 g, 1.372 mmol) and compound 158 (0.650 g, 2.095 mmol) was azeotroped twice with MeCN (20 mL each). To the resulting residue were added MeCN (20.0 ml) and 1H-imidazole-4,5-dicarbonitrile (0.243 g, 2.058 mmol). After complete reaction (monitored by LCMS), it was treated with (E)-N,N-dimethyl-N'-(3-thione-3H-1,2,4-dithiazol-5-yl)formamidine ( 0.422 g, 2.058 mmol) to work up the reaction mixture. After complete sulfurization, saturated _NaHCO3 solution (20 mL) and MTBE (30 mL) were added. The layers were separated and the aqueous layer was extracted with a MTBE/EtOAc mixture (15/15 mL). The combined organic layers were washed with 30% aqueous NaCl (10 mL), dried over _MgSO4 , filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography ( _SiO2 50 g, 33%-100% EtOAc in n-heptane plus 1% TEA) to give 0.88 g of compound 153.

LC/MS (ESI) m/z 1076.48 [M+Na] ⁺ .

Compound 154

To a solution of compound 153 (0.880 g, 0.835 mmol) in pyridine (8.8 ml) was added diphenyl phosphite (0.323 ml, 1.67 mmol) at ambient temperature. The reaction mixture was stirred for 1 hour and monitored by LCMS. Upon completion, the mixture was added to a mixture of saturated aqueous _NaHCO3 (13.2 ml) and water (4.4 ml) while keeping the internal temperature below 30°C, rinsing with EtOAc (8.8 ml). The resulting mixture was stirred at ambient temperature and hydrolysis was monitored by LCMS. After completion, the mixture was extracted twice with EtOAc/MTBE mixture (1/1, 26 mL). The combined organic layers were washed with 30% aqueous NaCl (3 ml), dried over _MgSO4 , filtered, and concentrated in vacuo. The residue was azeotroped twice with toluene (7 mL each). The crude product was dissolved in dichloromethane (6.6ml) and mixed with water (0.15ml, 8.35mmol) and 6% dichloroacetic acid (0.41ml, 5.0mmol) in dichloromethane (6.6ml) at ambient temperature deal with. After complete DMT deprotection (monitored by LCMS), triethylsilane (2.7 ml, 17 mmol) was added. After stirring for 10 minutes, the resulting mixture was treated with pyridine (4.4 ml) and TEA (1 ml) and concentrated in vacuo. The residue was triturated three times with n-heptane (8.8 ml each) and azeotroped twice with MeCN. The crude product (compound 154) was used in the next step without further purification.

LC/MS (ESI) m/z 814.29 [MH] ⁻ .

Compound 155

To a solution of compound 154 (0.681 g, 0.835 mmol) in pyridine (68 ml) was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphalane at ambient temperature 2-oxide (0.462 g, 2.505 mmol). The resulting solution was stirred at ambient temperature for 1 hour and monitored by LCMS. Upon completion, water (0.45 ml, 25 mmol) (10 equiv. DMOCP) and sulfur (0.134 g, 4.175 mmol) were added. After complete sulfurization (monitored by LCMS), the reaction mixture was treated with saturated aqueous _NaHCO3 (13.6 ml) and concentrated in vacuo. The residue was treated with EtOAc (27ml) and water (13.6ml). The layers were separated and the aqueous layer was extracted with EtOAc/MTBE mixture (1/1, 27 mL). The combined organic layers were washed with 30% aqueous NaCl (13.6 ml), dried over _MgSO4 , filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography ( _SiO2 25g, 0%-10% MeOH in EtOAc) to give 0.693g (theoretical yield) of compound 155.

LC/MS (ESI) m/z 830.23 [M+H] ⁺ .

Compound 156

To a solution of compound 155 (0.693 g, 0.835 mmol) in acetonitrile (14 ml) was added tert-butylamine (14 ml, 131 mmol) at ambient temperature. The resulting solution was stirred for 10 minutes and monitored by LCMS. Upon completion, the reaction mixture was concentrated in vacuo and azeotroped twice with MeCN. To the residue was added acetonitrile (14 ml) and 2-nitrobenzyl bromide (0.541 g, 2.51 mmol). After stirring overnight, the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography ( _SiO2 100 g, 75%-100% EtOAc in n-heptane and 0%-10% MeOH in EtOAc) to give 0.103 g of compound 156.

¹ H NMR (400MHz, chloroform-d) δ=8.59(s, 2H), 8.05(br d, J=8.2Hz, 2H), 8.03(s, 2H), 7.58(d, J=7.4Hz, 2H) ,7.48(t,J＝7.4Hz,2H),7.45-7.37(m,2H),6.20(br d,J＝18.8Hz,2H),6.21-6.08(m,2H),5.98-5.83(m, 2H), 5.69 (dd, J=4.7, 52.0Hz, 1H), 5.47 (br d, J=17.2Hz, 2H), 5.31 (br d, J=10.6Hz, 2H), 5.14 (br d, J= 5.5Hz, 4H), 4.54-4.46(m, 2H), 4.45-4.40(m, 2H), 4.37-4.26(m, 4H), 4.21-4.11(m, 2H).

Based on synthetic methodology, NMR data (symmetric), and HPLC retention time (slowest eluting isomer), Applicants believe that compound 156 has a RR phosphorous stereochemistry. This stereochemical assignment was confirmed by X-ray crystallography.

Compound 157

To a refluxing solution of compound 156 (0.103 g, 0.098 mmol) in toluene (103 ml) was added the second generation Hoveyda-Grubbs catalyst ((1,3-bis-(2,4,6-trimethylphenyl)- 2-imidazolidineidene)dichloro(o-isopropoxyphenylmethylene)ruthenium; can Obtained as Cat. No. 569755; CAS 301224-40-8; 31 mg, 0.049 mmol) and a solution of quinone (32 mg, 0.295 mmol) in toluene (10 mL). The mixture was stirred at reflux for 2 hours and additional catalyst (16 mg, 0.025 mmol) was added. Upon completion, the reaction mixture was cooled to ambient temperature and treated with DMSO (0.14 ml, 2.0 mmol) overnight. The resulting mixture was concentrated in vacuo and purified by silica gel column chromatography ( _SiO2 10 g, 0%-10% methanol in EtOAc) to give the desired product, which was further purified by pre TLC (EtOAc) to give 3.6 mg of compound 157 .

¹ H NMR (400 MHz, chloroform-d) δ=8.11 (dd, J=1.2, 8.2 Hz, 2H), 8.09 (s, 2H), 7.68 (s, 2H), 7.67-7.58 (m, 4H), 7.52 -7.48(m, 2H), 6.26(d, J=17.6Hz, 2H), 6.02-5.97(m, 2H), 5.97-5.88(m, 2H), 5.68(dd, J=3.9, 50.8Hz, 2H) ), 5.15 (br d, J=10.9Hz, 2H), 4.99 (br d, J=10.9Hz, 2H), 4.56-4.44 (m, 8H), 4.25 (br t, J=6.1Hz, 2H).

Compound 26

To compound 157 (3.6 mg, 3.5 μmol) was added 1,4-dioxane (0.11 mL), thiophenol (0.045 mL, 0.44 mmol) and TEA (0.054 mL, 0.39 mmol). The resulting mixture was stirred at ambient temperature while monitoring the reaction by LCMS. After complete conversion, water (0.5 mL) was added. The resulting mixture was extracted three times with a n-heptane/toluene mixture (1/1, 0.4 mL each), followed by toluene (0.3 mL). The aqueous layer was concentrated in vacuo and treated with water (0.5 mL). The resulting solid was filtered off and rinsed with water (0.5 mL). The combined filtrates were lyophilized to give 2.0 mg of the bisTEA salt compound 26 as a white foamy solid.

¹ H NMR (400MHz, methanol-d ₄ ) δ=8.67(s, 2H), 8.18(s, 2H), 6.41(d, J=14.8Hz, 2H), 6.07-6.01(m, 2H), 5.56( dd,J=3.1,51.2Hz,2H),5.48-5.41(m,2H),5.08(br d,J=12.1Hz,2H),4.99-4.88(m,2H),4.52(br d,J= 12.5Hz, 2H), 4.40 (br d, J=9.8Hz, 2H), 3.98 (dd, J=5.7, 12.3Hz, 2H), 3.14 (q, J=7.4Hz, 12H), 1.27 (t, J =7.4Hz, 18H).

Example 15--Synthesis of Compound 27

Compound 27 was prepared from compound 159, a by-product of stage 11b of the compound 1 pathway shown in Figure 2B.

To a solution of compound 159 (2.0 g, 1.75 mmol) in methanol (24 ml) was added 28% ammonium hydroxide (12 ml). The resulting mixture was stirred at 50°C for 5 hours and cooled to ambient temperature. After completion (monitored by LCMS), the reaction mixture was cooled to ambient temperature, treated with water (20 mL), and extracted three times with a toluene/EtOAc mixture (1/1, 24 mL each). The aqueous layer was acidified with 1.0M hydrochloric acid (3.5ml, 3.5mmol) and stirred at ambient temperature for 30 minutes and at 0°C for 30 minutes. The resulting slurry was filtered and rinsed with water (20 mL). The filter cake was dried in a vacuum oven at 35°C overnight and dissolved with a mixture of 28% _NH4OH (2 mL) and MeOH (20 mL). The resulting solution was concentrated in vacuo and slurried with EtOH (4 ml). The resulting solid was collected by filtration and dried in vacuo. 70 mg of compound 27 was obtained as a white solid.

¹ H NMR (400MHz, methanol-d ₄ )δ=9.03(br s,1H),8.31(br s,1H),8.26(br s,1H),8.13(br s,1H),6.43-6.24(m ,2H),5.97-5.83(m,2H),5.71(br d,J=51.6Hz,1H),5.64(brd,J=50.0Hz,1H),5.12-4.99(m,1H),4.96-4.90 (m, 1H), 4.68-4.32 (m, 6H), 4.14-3.97 (m, 2H), 3.77-3.62 (m, 2H).

Example 16--Synthesis of Compound 28

Compound 28 was prepared using the same method as compound 27 from compound 160, the product of stage 11 of the compound 1 pathway shown in Figure 2B.

¹ H NMR (400MHz, methanol-d ₄ )δ=8.70(br s,1H),8.49(br s,1H),8.24(br s,1H),8.10(br s,1H),6.52-6.26(m ,2H),5.94-5.78(m,2H),5.69(br d,J＝54.7Hz,1H),5.48-5.26(m,1H),5.14(br d,J＝53.1Hz,2H),5.06- 4.94(m,1H),4.74-4.19(m,4H),4.10-3.93(m,2H),3.76-3.58(m,2H)

Example 17--Synthesis of Compound 29

Compound 162

Compound 161 (5.0 g, 6.329 mmol) and but-2-yn-1,4-diol (1.907 g, 22.153 mmol) were azeotroped twice with THF. The residue was dissolved in THF (50.0 ml) and toluene (75 ml). Triphenylphosphine (2.158 g, 8.228 mmol) was added and the resulting solution was cooled to below 5°C. DIAD (1.60 ml, 8.23 mmol) was added dropwise while keeping the internal temperature below 10°C. The resulting solution was stirred at 0-5°C while progress was monitored by LCMS. Upon completion, the reaction mixture was concentrated in vacuo to remove most of the THF. The residue was diluted with MTBE (50.0 ml) and washed twice with 30% aqueous NaCl (40.0 ml each) and twice with water (25 ml each). The organic solution was concentrated in vacuo and purified by silica gel column chromatography ( _SiO2 100 g, 50%-70% EtOAc in n-heptane buffered with 1% TEA) to afford 4.671 g of compound 162.

¹ H NMR (400 MHz, chloroform-d) δ=8.61 (s, 1H), 8.14 (s, 1H), 7.43 (d, J=7.2 Hz, 2H), 7.33-7.27 (m, 3H), 7.24-7.18 (m, 8H), 7.05-7.01 (m, 2H), 6.76 (d, J=8.6Hz, 4H), 6.20 (dd, J=2.7, 17.2Hz, 1H), 5.59 (td, J=3.1, 53.1 Hz, 1H), 5.19 (br d, J=2.3Hz, 2H), 4.93-4.83 (m, 1H), 4.18-4.15 (m, 1H), 4.01 (br d, J=6.3Hz, 2H), 3.78 (s,6H),3.53(dd,J=2.7,10.9Hz,1H),3.17(dd,J=3.7,11.1Hz,1H),0.85(s,9H),0.10(s,3H),0.02( s, 3H).

Compound 164

Compound 162 (4.671 g, 5.444 mmol), compound 163 (3.32 g, 6.805 mmol) and triphenylphosphine (1.856 g, 7.077 mmol) were dissolved in THF (56.1 ml) and cooled to below 5°C. To the resulting solution was added DIAD (1.3 ml, 6.8 mmol) while keeping the internal temperature below 10°C. After complete consumption of compound 162 (monitored by LCMS), the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography ( _SiO2 340 g, 55-70% EtOAc in n-heptane buffered with 1% TEA) to give 3.879 g Compound 164 as a white foamy solid.

¹ H NMR (400MHz, chloroform-d) δ=8.51(s, 1H), 8.45(s, 1H), 8.20(s, 1H), 8.05(s, 1H), 7.41(d, J=7.1Hz, 2H ),7.36-7.32(m,4H),7.28-7.13(m,8H),6.98(t,J=7.8Hz,2H),6.79-6.74(m,4H),6.29(dd,J=2.0,16.4 Hz,1H),6.19(dd,J＝2.7,17.2Hz,1H),5.53(ddd,J＝2.7,4.7,53.1Hz,1H),5.33(ddd,J＝2.0,3.9,53.1Hz,1H) ,5.23-5.21(m,1H),5.05-5.01(m,2H),4.99-4.94(m,2H),4.81(ddd,J=4.3,6.7,16.7Hz,1H),4.71-4.60(m, 1H), 4.21-4.14(m, 2H), 4.08-4.03(m, 1H), 3.89(dd, J=3.1, 11.7Hz, 1H), 3.77(s, 6H), 0.91(s, 9H), 0.84 (s, 9H), 0.09 (s, 3H), 0.08 (s, 6H), 0.00 (s, 3H).

Compound 165

To a solution of compound 164 (1.5 g, 1.13 mmol) in pyridine (12.0 ml) was added diphenyl phosphite (0.35 ml, 1.8 mmol) at ambient temperature. The resulting solution was stirred at ambient temperature while being monitored by LCMS. Upon completion, the reaction mixture was added to a mixture of saturated aqueous NaHCO ₃ (22.5 ml) and water (7.5 ml) while keeping the internal temperature below 30°C. After the hydrolysis was complete (monitored by LCMS), the resulting mixture was extracted twice with EtOAc/MTBE mixture (30.0/30.0 ml). The combined organic layers were washed with 30% aqueous NaCl (15.0 ml), dried over _MgSO4 , filtered, and concentrated in vacuo. The residue was azeotroped three times with toluene and dissolved in dichloromethane (11 ml). Work up with water (0.20 ml, 11 mmol) and 6% dichloroacetic acid (0.57 ml, 6.9 mmol) in dichloromethane (11 ml) at ambient temperature. After complete removal of the DMT group (monitored by LCMS), triethylsilane (1.8 ml, 11 mmol) was added. After stirring for 10 minutes, TEA (1.6 ml, 11 mmol) was added and the resulting mixture was concentrated in vacuo. The residue was dissolved in EtOAc (22.5ml), washed with water (3.8ml) and saturated aqueous _NaHCO3 (8%) (3.0ml), dried over _MgSO4 , filtered, and concentrated in vacuo. The crude product was triturated twice with n-heptane (33.8 ml) and a mixture of n-heptane (9.0 ml) and toluene (3.0 ml). The supernatant was decanted and the solid remaining at the bottom was dissolved in acetonitrile. The resulting solution was concentrated in vacuo and azeotroped twice with acetonitrile. The crude product compound 165 was used in the next stage without purification (assuming 100% theoretical yield).

LC/MS (ESI) m/z 1089.74 [M+H] ⁺ .

Compound 166

To a solution of compound 165 (1.231 g, 1.13 mmol) in pyridine (100 mL) was added TEA (0.47 ml, 3.4 mmol) and DMOCP (0.375 mg, 2.03 mml) at ambient temperature. The resulting solution was stirred at ambient temperature while being monitored by LCMS. Upon completion, water (0.41 mL, 22.6 mmol) was added followed by sulfur (0.181 g, 5.651 mmol). After complete sulfurization (monitored by LCMS), saturated aqueous _NaHCO3 (8%) (24.6 ml) and water (10 mL) were added. The resulting mixture was concentrated in vacuo to about 50 mL and extracted twice with a MTBE/EtOAc mixture (31/31 mL each). The combined organic layers were washed with 30% aqueous NaCl (25 ml), dried over _MgSO4 , filtered, concentrated in vacuo, and subjected to column chromatography on silica gel ( _SiO2 100 g, 0-10% methanol in ethyl acetate buffered with 1% TEA) ) was purified to give compound 166 (0.484 g).

LC/MS (ESI) m/z 1103.65 [M+H] ⁺ .

Compound 167

To a solution of compound 166 (0.484 g, 0.402 mmol) in pyridine (1.2 ml) and TEA (5.8 ml) was added _Et3N3HF (0.581 ml, 3.563 mmol) at ambient temperature. The resulting mixture was stirred at ambient temperature while being monitored by LCMS. After complete conversion, methoxytrimethylsilane (3.9 ml, 28 mmol) was added. After stirring for 20 minutes at ambient temperature, the supernatant was decanted. Toluene (5 mL) was added to the residue, and after stirring for 10 minutes, the toluene layer was decanted. The same operation is repeated again. The resulting crude product was dissolved in dichloromethane (10 mL), washed with saturated aqueous _NH4Cl (27 wt%) (5 ml) and 30% aqueous NaCl (2.4 ml), and dried over _MgSO4 . The resulting organic layer was concentrated after filtration to give 0.386 g of a light brown solid, which was azeotroped twice with MeCN. The resulting crude product was dissolved in MeCN (4.6 ml) and treated with 2-nitrobenzyl bromide (0.103 g, 0.475 mmol) at ambient temperature. After the alkylation was complete (monitored by LCMS), the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography ( _SiO2 25 g, 0-5% MeOH in EtOAc) to afford 0.251 g of compound 167 as a white solid.

¹ H NMR (400MHz, chloroform-d)δ=8.42(br s,1H),8.31(br s,1H),8.22(brs,1H),8.10-8.04(m,1H),7.93(s,1H) ,7.53-7.37(m,8H),7.32-7.27(m,3H),7.24-7.16(m,2H),6.18(br d,J=16.8Hz,1H),6.26-6.14(m,1H), 5.56(d,J＝53.1Hz,1H),5.48-5.37(m,1H),5.31(br s,1H),5.15(br d,J＝17.6Hz,1H),4.92(d,J＝17.6Hz ,1H),4.82(d,J＝17.2Hz,1H),4.88-4.77(m,1H),4.76-4.66(m,1H),4.58-4.33(m,3H),4.29(br d,J＝ 5.9Hz, 1H), 4.21 (br s, 1H), 4.13-4.06 (m, 1H), 3.93-3.79 (m, 2H).

Compound 168

Compound 167 (0.224 g, 0.222 mmol) and 3-(bis(diisopropylamino)phosphinooxy)propionitrile (0.081 ml, 0.255 mmol) were dissolved in MeCN (5 mL) and concentrated. The same operation is repeated two more times. The resulting mixture was dissolved in dichloromethane (2.2 ml), cooled to 0 °C, and treated with diisopropylammonium tetrazolium (0.019 g, 0.111 mmol). The reaction mixture was allowed to warm to ambient temperature overnight and diluted with MeCN (2.2 mL). The resulting solution was added via a syringe pump to a solution of pyridine trifluoroacetate (0.129 g, 0.665 mmol) in MeCN (18 ml) over 10 hours. After stirring for an additional hour, 3-(bis(diisopropylamino)phosphino)propionitrile (0.04 mL, 0.12 mmol) in MeCN (1 mL) was added over 4 hours. (E)-N,N-Dimethyl-N'-(3-thione-3H-1,2,4-dithiazol-5-yl)formamidine (0.064 g, 0.311 mmol) was added. After complete sulfurization (monitored by LCMS), the reaction mixture was concentrated in vacuo and the resulting residue was dissolved in MTBE (4.5 ml). The resulting organic solution was washed with saturated aqueous _NaHCO3 (8%) (4 ml) and water (2 ml). The combined aqueous layers were back extracted with a MTBE/EtOAc mixture (4/4 mL). The combined organic layers were washed twice with 30% aqueous NaCl (2 mL each), dried over _MgSO4 , filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography ( _SiO2 25g, 50%-100% EtOAc in n-heptane) to give 64 mg of compound 168.

¹ H NMR (400MHz, chloroform-d) δ=8.57(s,1H), 8.18(s,1H), 8.06-8.01(m,1H), 7.91(s,1H), 7.62-7.56(m,2H) ,7.55-7.50(m,2H),7.49-7.43(m,3H),7.42-7.34(m,2H),7.30-7.18(m,5H),6.33(d,J=15.2Hz,1H),6.10 (d, J=20.7Hz, 1H), 5.95-5.77 (m, 1H), 5.54 (dd, J=4.3, 52.8Hz, 1H), 5.46 (dd, J=3.9, 50.8Hz, 1H), 5.32- 5.18(m, 1H), 5.10(d, J=17.6Hz, 1H), 4.89(d, J=17.6Hz, 1H), 4.85-4.81(m, 2H), 4.78(br d, J=12.1Hz, 1H), 4.64(br dd, J=4.1, 9.2Hz, 1H), 4.55(dd, J=2.7, 12.1Hz, 1H), 4.52-4.44(m, 3H), 4.38-4.20(m, 4H), 2.75 (td, J=6.3, 17.6 Hz, 1H), 2.58 (td, J=5.9, 17.2 Hz, 1H).

Compound 29

To a solution of compound 168 (40 mg, 0.035 mmol) in 1,4-dioxane (0.5 mL) was added thiophenol (0.24 ml, 2.3 mmol) and TEA (0.24 ml, 1.7 mmol). The resulting mixture was stirred at ambient temperature while monitoring the reaction by LCMS. Upon completion, methanol (0.64ml) and 28% ammonium hydroxide (0.64ml) were added. The resulting mixture was heated to 50°C, stirred for 5 hours, and cooled to ambient temperature. After complete deprotection (monitored by LCMS), the resulting mixture was treated with water (0.80 ml) and extracted three times with a n-heptane/toluene mixture (1/1, 0.5 mL each) followed by toluene (0.3 ml). The aqueous layer was concentrated in vacuo at 40-50°C and treated with water (1 mL). The resulting solid was filtered off and rinsed with water (0.3 ml). The combined filtrates were treated with 1.0M HCl (0.07ml, 0.07mmol). The resulting slurry was filtered and rinsed with water (1 mL). The filter cake was dissolved with 2.0M ammonia solution in MeOH (1.0 ml, 2.0 mmol). The resulting solution was concentrated in vacuo to 0.5 mL and treated with EtOH (0.5 mL). The same operation is repeated two more times. To the resulting solution was added EtOAc (0.9 mL) dropwise. Precipitation occurs. The resulting solid was collected by filtration, rinsed with a 4/1 mixture of EtOAc/EtOH (0.4 mL), and dried in vacuo at ambient temperature overnight. 12 mg of compound 29 were obtained.

¹ H NMR (400MHz, methanol-d ₄ )δ=8.88(br s,1H),8.50(br s,1H),8.20(br s,1H),8.12(br s,1H),6.42-6.17(m ,2H),5.84-5.54(m,1H),5.52(d,J=51.6Hz,1H),5.06-4.78(m,2H),4.77-4.55(m,3H),4.49(br d,J= 10.2Hz, 1H), 4.42 (br s, 2H), 4.09-3.77 (m, 4H).

Example 18--Synthesis of Compound 25

Compound 170

To a refluxing solution of second generation Hoveyda-Grubbs catalyst (0.996 g, 1.585 mmol) in dichloromethane (40.0 ml) was added prop-2-en-d5-1-ol (10.0 g, 158 mmol). After 1 hour, an additional 1 mol% of the second generation Hoveyda-Grubbs catalyst (0.996 g, 1.585 mmol) was added. The resulting solution was stirred at reflux overnight, cooled to ambient temperature, and concentrated in vacuo. The residue was purified by silica gel column chromatography ( _SiO2 340 g, 0%-10% MeOH in EtOAc) to give 4.2 g of compound 170 as a brown oil.

¹³ C NMR (101 MHz, methanol-d ₄ ) δ=130.87 (t, J=23.8 Hz, 2C), 62.22 (quin, J=21.9 Hz, 2C).

Compound 171

Using compound 170 as starting material, compound 171 was prepared by the same reaction sequence (stages 1-11) as described in the synthetic route of compound 1 in Figures 2A and 2B.

¹ H NMR (400MHz, chloroform-d) δ=8.59(s,1H), 8.10(s,1H), 8.02-7.96(m,1H), 7.93(s,1H), 7.58-7.50(m,2H) ,7.48-7.37(m,5H),7.34-7.27(m,2H),7.24-7.14(m,4H),6.90(s,1H),6.28(d,J＝14.8Hz,1H),6.14-6.03 (m,1H),5.97-5.81(m,1H),5.49(dd,J=4.7,52.3Hz,2H),5.43(dd,J=3.9,50.8Hz,1H),5.30-5.15(m,1H) ),4.75(br d,J=12.1Hz,1H),4.61(br dd,J=4.3,9.4Hz,1H),4.53(dd,J=2.9,11.9Hz,1H),4.47-4.41(m, 3H), 4.32-4.18(m, 4H), 2.79(td, J=7.0, 17.2Hz, 1H), 2.66(td, J=6.3, 16.8Hz, 1H)

Compound 25

Compound 25 was prepared by the same reaction sequence (stages 12-13) as described in Figures 2A and 2B using compound 171 as starting material.

¹ H NMR (400MHz, CD ₃ OD)δ=9.03(br s,1H),8.30(br s,1H),8.24(br s,1H),8.10(br s,1H),6.44-6.13(m, 2H), 0.00(d, J＝52.4Hz, 1H), 0.00(d, J＝51.2Hz, 1H), 4.71-4.32(m, 6H), 4.09-3.98(m, 1H), 3.97-3.87(m , 1H).

Example 19--Synthesis of compound 31 (RpRp or SpSp)

Compound 173

A mixture of (E)-but-2-ene-1,4-diol (0.10 g, 1.135 mmol) and compound 172 (2.52 g, 2.55 mmol) was azeotroped twice with THF and dissolved in THF (5.0 ml) ) and toluene (7.5 ml). To the resulting solution was added triphenylphosphine (0.714 g, 2.724 mmol). The resulting solution was cooled to below 5°C and treated with DIAD (0.53 ml, 2.72 mmol) while keeping the internal temperature below 10°C. The resulting reaction mixture was stirred overnight while warming to ambient temperature. After complete reaction (monitored by LCMS), the reaction mixture was concentrated in vacuo and passed through silica gel column chromatography ( _SiO2 50 g, pretreated with 1% TEA in n-heptane/EtOAc (1/1), 50%-66% EtOAc) to give 2.46 g of compound 173 as a white foamy solid. This material was used in the next step without further purification.

Compound 174

To a solution of compound 173 (2.387 g, 1.177 mmol) in acetonitrile (28.6 ml) was added water (0.085 ml, 4.7 mmol), pyridine trifluoroacetate (0.500 g, 2.589 mmol) at ambient temperature. After 1 minute, 2-amino-2-methylpropane (19.1 ml, 182 mmol) was added. The resulting mixture was stirred at ambient temperature while monitoring the reaction by LCMS. Upon completion, the reaction mixture was concentrated in vacuo and azeotroped with MeCN. The residue was dissolved in dichloromethane (24 mL) and treated with water (0.42 mL) and 6% dichloroacetic acid (1.2 mL, 14 mmol) in dichloromethane (24 mL). After complete DMT deprotection (monitored by LCMS), the reaction was quenched with pyridine (12 ml) and the reaction mixture was concentrated in vacuo. The resulting residue was treated with a n-heptane/toluene mixture (15/15 ml) and the top layer was decanted. The same operation is repeated again. The remaining residue was dried in vacuo to give compound 174, which was used in the next step without further purification.

LC/MS (ESI) m/z 1151.64 [M+H] ⁺ .

Compound 175

To a solution of compound 174 (1.355 g, 1.177 mmol) in pyridine (271 ml) was added TEA (0.98 ml, 7.1 mmol) and 2-chloro-5,5-dimethyl-1,3,3 at ambient temperature 2-Dioxaphosphalane 2-oxide (0.869 g, 4.708 mmol). The resulting solution was stirred at ambient temperature until all starting material was consumed (monitored by LCMS). After complete reaction, water (0.85 ml, 47 mmol) (10 equiv. DMOCP) was added followed by sulfur (0.377 g, 11.77 mmol). The resulting mixture was stirred at ambient temperature while monitoring the reaction by LCMS. After complete sulfurization, the reaction mixture was treated with saturated aqueous _NaHCO3 (27 mL) and water (10 mL), and concentrated in vacuo. To the resulting residue was added a mixture of MTBE/EtOAc (34/34 ml), saturated aqueous _NaHCO3 (27 ml) and water (20 mL). The layers were separated and the aqueous layer was extracted with a mixture of EtOAc/MTBE (34/34 mL). The combined organic layers were washed with 30% aqueous NaCl (7 ml), dried over _MgSO4 , filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography ( _SiO2 50 g, 0%-20% MeOH in EtOAc plus 1% TEA) to give 56 mg of compound 175 (symmetric based on NMR; RpRp or SpSp isomers).

¹ H NMR (400MHz, chloroform-d) δ=9.09(s, 2H), 8.46(s, 2H), 7.56-7.05(m, 10H), 6.37(d, J=8.2Hz, 2H), 6.28-6.22 (m,2H),5.29(ddd,J=4.7,7.8,11.7Hz,2H),5.00(br d,J=13.7Hz,2H),4.84(d,J=4.3Hz,2H),4.76(br d, J＝13.3Hz, 2H), 4.31(br s, 2H), 4.28-4.20(m, 2H), 4.16-4.08(m, 2H), 3.00-2.77(m, 12H), 1.12-0.99(m , 18H), 0.96(s, 18H), 0.34(s, 6H), 0.25(s, 6H).

Compound 31

To a solution of compound 175 (56 mg, 0.0405 mmol) in methanol (1.1 mL) was added 28% ammonium hydroxide (0.22 mL). The resulting mixture was stirred at 50°C for 5 hours, cooled to ambient temperature, concentrated in vacuo, and azeotroped twice with MeCN. To the resulting residue was added pyridine (0.22 mL), TEA (0.11 mL) and TEA 3HF (0.11 ml, 0.69 mmol). The resulting mixture was stirred at 50 °C for 5 hours, cooled to ambient temperature, and treated with methoxytrimethylsilane (0.90 mL, 6.50 mmol) for 30 minutes. The resulting mixture was concentrated in vacuo and dissolved in water (5 mL). The resulting aqueous solution was extracted three times with toluene (5 mL each) and concentrated in vacuo. The crude product was dissolved in 1 mL of water, filtered through a syringe filter, and acidified with 1 N HCl to pH <3. The resulting mixture was kept in a refrigerator (5°C) for 2 days and the resulting solid was collected by filtration. The solid was dissolved in 2M _NH3 in MeOH (1.5 ml) and concentrated in vacuo to give 2.8 mg of compound 31 as a mixture of _NH3 and TEA salt. The product was dissolved in EtOH (1 mL), treated with 0.1 ml TEA, concentrated to 0.5 mL, and stored in a refrigerator (-20°C) overnight. The supernatant was removed and the remaining solid was further dried in vacuo to yield 0.8 mg of the bisTEA salt compound 31 (symmetric by NMR; RpRp or SpSp isomer).

¹ H NMR (400MHz, methanol-d ₄ )δ=8.26-7.83(m,4H),6.37(br s,2H),5.89(br s,2H),5.36-5.07(m,2H),4.95(br d, J=3.5Hz, 2H), 4.58 (br dd, J=6.3, 10.6Hz, 2H), 4.43 (brs, 2H), 4.12-4.00 (m, 4H), 3.89-3.70 (m, 2H), 3.19(q,J=7.2Hz,12H),1.30(t,J=7.4Hz,18H)

Example 20--Synthesis of compound 32

Compound 178

To compound 177 (2.0 g, 2.532 mmol) and (+)-2,3-O-isopropylidene-L-threitol (compound 176) (1.232 g, 7.595 mmol) in THF (20.0 ml) and toluene To the solution in (30.0 ml) was added triphenylphosphine (0.930 g, 3.544 mmol). The resulting solution was cooled to below 5°C and treated with DIAD (0.64 ml, 3.3 mmol). The reaction mixture was warmed to ambient temperature. After stirring at ambient temperature for 8 hours, additional DIAD (0.64 mL) and _PPh3 (0.93 g) were added. After stirring overnight at ambient temperature, the reaction mixture was stirred at 40°C for 2 days and concentrated in vacuo. The residue was purified by silica gel column chromatography ( _SiO2 100 g, pretreated with 1% TEA in n-heptane/EtOAc (1/1), 50%-70% EtOAc in n-heptane) to give 0.524 g Compound 178 as a white foamy solid.

¹ H NMR (400MHz, chloroform-d) δ=8.62(s, 1H), 8.09(s, 1H), 7.40-7.09(m, 12H), 7.01-6.92(m, 2H), 6.80-6.73(m, 4H), 6.16 (dd, J=2.7, 16.8Hz, 1H), 5.49 (ddd, J=3.1, 4.3, 53.1Hz, 1H), 4.85-4.75 (m, 2H), 4.63 (dd, J=3.9, 14.8Hz, 1H), 4.30-4.19 (m, 2H), 4.18-4.12 (m, 1H), 3.94 (tt, J=3.5, 8.2Hz, 1H), 3.84 (td, J=5.5, 12.1Hz, 1H) ),3.78(s,6H),3.53(dd,J＝2.7,10.9Hz,1H),3.17(dd,J＝3.5,10.9Hz,1H),2.94(br t,J＝5.5Hz,1H), 0.89(s,6H),0.83(s,9H),0.08(s,3H),-0.01(s,3H)

Compound 180

To a solution of compound 178 (0.409 g, 0.84 mmol) in THF (6 mL) was added triphenylphosphine (0.191 g, 0.728 mmol) and DIAD (0.142 ml, 0.728 mmol). After stirring at ambient temperature for 40 hours, the reaction mixture was concentrated in vacuo and passed through column chromatography ( _SiO2 50 g, pretreated with 1% TEA in n-heptane/EtOAc (1/1), 50 in n-heptane %-66% EtOAc) to give 0.533 g of compound 180.

LC/MS: LRMS (ESI) m/z 1403.86 [M+H] ⁺ .

Compound 181

Compound 181 was prepared by the same reaction sequence (stages 5-11) as described in Figures 2A and 2B using compound 180 as starting material.

¹ H NMR (400MHz, chloroform-d) δ=8.72(s,1H), 8.14-8.06(m,1H), 7.93(s,1H), 7.69(s,1H), 7.62-7.53(m,6H) ,7.53-7.45(m,1H),7.43-7.33(m,2H),7.30-7.24(m,3H),7.19-7.12(m,2H),6.11(d,J=16.8Hz,1H),6.05 (d, J=19.5Hz, 1H), 5.89 (dd, J=3.5, 52.3Hz, 1H), 5.57-5.46 (m, 1H), 5.41 (dd, J=3.5, 53.1Hz, 1H), 5.18- 5.04(m,1H),4.83(dd,J=4.7,14.4Hz,1H),4.78-4.73(m,1H),4.70(br d,J=12.1Hz,1H),4.64-4.48(m,4H ), 4.46-4.35(m, 4H), 4.33-4.27(m, 2H), 4.16-4.06(m, 2H), 4.05-3.96(m, 1H), 2.55(t, J=6.1Hz, 2H), 1.26(s, 6H).

Compound 182

To a solution of compound 181 (3.2 mg, 2.629 μmol) in dichloromethane (0.5 ml) was added tert-butylamine (0.5 ml, 4.7 mmol). After stirring for 30 minutes, the reaction solution was concentrated in vacuo and azeotroped twice with MeCN. The residue was dissolved in acetonitrile (1 ml) and treated with 1-(bromomethyl)-2-nitrobenzene (1.7 mg, 7.9 μmol). After complete alkylation (monitored by LCMS), the reaction mixture was concentrated under a nitrogen purge. The residue was purified by silica gel column chromatography ( _SiO2 4g, 80%-100% EtOAc in n-heptane) to give 2 mg of compound 182.

¹ H NMR (400MHz, chloroform-d) δ=8.12 (dd, J=1.4, 8.0Hz, 1H), 8.03-8.00 (m, 1H), 8.00 (s, 1H), 7.93 (s, 1H), 7.70 (s,1H),7.70-7.29(m,13H),7.21(dt,J=2.7,7.6Hz,4H),6.07(d,J=19.9Hz,1H),5.94(d,J=21.1Hz, 1H),5.91-5.77(m,1H),5.81(dd,J=4.3,51.5Hz,1H),5.62-5.49(m,1H),5.54(dd,J=3.1,52.7Hz,2H),4.83 (q, J=5.9 Hz, 1H), 4.72-4.63 (m, 2H), 4.61-4.28 (m, 13H), 1.42 (s, 3H), 1.28 (s, 3H).

Compound 32

To compound 182 (2.0 mg, 1.5 μmol) was added acetic acid (0.8 ml) and water (0.2 ml). The resulting mixture was stirred at ambient temperature for 14 hours, at 45-50°C for 24 hours, concentrated in vacuo, and azeotroped twice with toluene. To the residue were added 1,4-dioxane (0.12 mL), thiophenol (60 μl) followed by TEA (60 μl). The resulting mixture was stirred at ambient temperature while monitoring the reaction by LCMS. Upon completion, methanol (160 μl) and 28% ammonium hydroxide (160 μl) were added. The resulting mixture was stirred at 50°C until debenzoylation was complete (monitored by LCMS). Upon completion, water (0.3 ml) was added. The resulting solid was filtered off and rinsed with water (0.2 mL). The filtrate was extracted twice with toluene (0.5 mL each) and concentrated in vacuo at 40-50°C. The residue was treated with water (0.5 mL) and the resulting solid was filtered off and rinsed with water (0.1 mL). The combined aqueous layers were subjected to HPLC purification under the following conditions to yield 1.5 mg of compound 32.

LC/MS: LRMS (ESI) m/z 781.23 [M+H] ⁺ .

Preparative HPLC conditions for compound 32:

Example 21--Synthesis of Compound 33 and Compound 34

At 0 °C, to 9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,9-dihydro-6H- Purin-6-one, compound 183 (5.00 g, 18.5 mmol) in pyridine (75 ml) was added _Ac2O (7.0 ml, 74 mmol) and DMAP (0.565 g, 4.626 mmol). The resulting mixture was warmed to ambient temperature and stirred while monitoring the reaction by LCMS. Upon completion, the reaction mixture was concentrated in vacuo and treated with EtOAc (200 mL) and water (50 mL). Precipitation occurs. The resulting solid was collected by filtration and rinsed with MTBE. Vacuum drying at 40°C overnight afforded 5.81 g of compound 193 as a white solid.

¹ H NMR (400MHz, DMSO-d ₆ ) δ=12.53 (br s, 1H), 8.33 (s, 1H), 8.15 (d, J=4.3 Hz, 1H), 6.37 (dd, J=2.7, 19.1 Hz ,1H),5.86(ddd,J＝2.7,5.1,51.6Hz,1H),5.62(ddd,J＝5.5,7.0,16.0Hz,1H),4.49-4.38(m,2H),4.26(dd,J = 4.7, 12.1 Hz, 1H), 2.19 (s, 3H), 2.04 (s, 3H).

Compound 184

To a solution of compound 193 in dichloromethane (40.0 ml) at 0°C was slowly added DMF (1.3 ml, 16.9 mmol) and thionyl chloride (1.28 ml, 17.5 mmol). The resulting mixture was heated to reflux and stirred until all starting material was consumed (monitored by LCMS). Upon completion, the reaction mixture was cooled to ambient temperature and treated with saturated aqueous _NaHCO3 (40 mL). The layers were separated and the aqueous layer was extracted twice with dichloromethane (30 mL each). The combined organic layers were dried over _MgSO4 , filtered, and concentrated in vacuo to give 2.81 g of compound 184 as a light brown oil.

¹ H NMR (400 MHz, chloroform-d) δ=8.79 (s, 1H), 8.30 (s, 1H), 6.29 (dd, J=2.0, 18.0 Hz, 1H), 5.79 (ddd, J=1.6, 4.7, 51.6Hz, 1H), 5.53 (ddd, J=5.1, 7.6, 17.8Hz, 1H), 4.58-4.48 (m, 2H), 4.33 (dd, J=3.9, 12.5Hz, 1H), 2.20 (s, 3H) ), 2.07(s, 3H).

Compound 194

To a solution of compound 184 (0.22 g, 0.59 mmol) in a mixture of THF (7.7 ml) and NMP (0.77 ml) at ambient temperature was added iron(III) acetylacetonate (0.021 g, 0.059 mmol) and a solution in THF of 0.5M 3-butenylmagnesium bromide (1.77 ml, 0.885 mmol). After complete reaction (monitored by LCMS), MTBE (10 mL) and 0.1 N HCl (10 mL) were added. The resulting mixture was stirred at ambient temperature for 10 minutes. The layers were separated and the aqueous layer was extracted with EtOAc/MTBE mixture (1/1, 10 ml). The combined organic layers were washed with 30% aqueous NaCl (5 mL), dried over _MgSO4 , and concentrated in vacuo. The crude product was used in the next step without further purification.

¹ H NMR (400MHz, chloroform-d) δ=8.90 (s, 1H), 8.16 (s, 1H), 6.26 (dd, J=2.0, 18.4Hz, 1H), 5.98-5.85 (m, 1H), 5.90 -5.73(m,1H),5.63(ddd,J=4.9,7.5,17.7Hz,1H),5.12-5.05(m,1H),4.98(dd,J=1.6,10.2Hz,1H),4.54-4.50 (m, 2H), 4.31(dd, J=5.1, 12.9Hz, 1H), 3.30(t, J=7.4Hz, 2H), 2.70-2.64(m, 2H), 2.19(s, 3H), 2.05( s, 3H).

Compound 185

The crude compound 194 (0.232 g, 0.590 mmol theoretical) was dissolved in methanol (1.5 ml) and treated with 2.0 M ammonia in MeOH (1.5 ml, 3.0 mmol) at ambient temperature. After complete deacetylation, the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography ( _SiO2 10 g, 0%-5% MeOH in EtOAc) to give 0.17 g of compound 185.

LCMS: MS(ESI) m/z 309.20 [M+H] ⁺

Compound 195

To a solution of compound 185 (1.22 g, 2.928 mmol) in pyridine (9.0 ml) at 0 °C was added 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (1.042 g, 3.075 mmol). The reaction mixture was warmed to ambient temperature while monitoring the reaction by LCMS. Upon completion, MTBE (18 mL) and saturated aqueous _NaHCO3 (9.0 mL) were added. After stirring for 10 minutes, the layers were separated and the aqueous layer was extracted with MTBE (18.0 ml). The combined organic layers were washed with 30% aqueous NaCl (10 ml), filtered, and concentrated in vacuo. Purification by silica gel column chromatography ( _SiO2 50 g, 50%-100% ethyl acetate in heptane plus 1% TEA) afforded 1.53 g of compound 195 as an orange solid.

¹ H NMR (400MHz, chloroform-d) δ=8.86-8.78(m, 1H), 8.21(s, 1H), 7.40-7.36(m, 2H), 7.31-7.18(m, 7H), 6.79(d, J=8.6Hz, 4H), 6.29 (dd, J=2.3, 17.2Hz, 1H), 5.92 (tdd, J=6.4, 10.4, 17.0Hz, 1H), 5.70 (ddd, J=2.7, 4.7, 52.8Hz) ,1H),5.09(dd,J＝1.6,17.2Hz,1H),4.97(dd,J＝1.6,10.2Hz,1H),4.90-4.77(m,1H),4.26-4.19(m,1H), 3.78(s, 6H), 3.56(dd, J=3.1, 10.9Hz, 1H), 3.43(dd, J=4.3, 10.9Hz, 1H), 3.33-3.28(m, 2H), 2.70-2.64(m, 2H), 2.20 (dd, J=2.5, 7.2Hz, 1H).

Compound 186

To a solution of compound 195 (1.530 g, 2.505 mmol) in dichloromethane (23 ml) was added 3-(bis(diisopropylamino)phosphinooxy)propionitrile (1.20 ml, 3.76 mmol) at ambient temperature ) and diisopropylammonium tetrazolium (0.493 g, 2.881 mmol). The reaction mixture was stirred at ambient temperature while progress was monitored by LCMS. Upon completion, the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography ( _SiO2 50 g, 40%-66% EtOAc in n-heptane plus 1% TEA) to afford 1.80 g of compound 186 as a light orange foamy solid.

Compounds 196 and 197

Compound 186 (1.8 g, 2.22 mmol) and compound 185 (1.110 g, 2.664 mmol) were dissolved in acetonitrile (21.6 ml) and concentrated in vacuo. The same operation is repeated again. The resulting mixture was dissolved in acetonitrile (21.6 ml) and treated with pyridinium trifluoroacetate (0.514 g, 2.664 mmol) at ambient temperature. After complete reaction (monitored by LCMS), ((dimethylamino-methylene)amino)-3H-1,2,4-dithiazoline-3-thione (0.911 g, 4.439 mmol) was added and added in The resulting mixture was stirred at ambient temperature. After complete sulfurization (monitored by LCMS), saturated aqueous _NaHCO3 (30 mL) was added and the resulting mixture was extracted three times with MTBE (30 mL each). The combined organic layers were washed with 30% aqueous NaCl (20 ml), dried over _MgSO4 , filtered, and concentrated in vacuo. Purification by silica gel column chromatography ( _SiO2 100 g, 0%-10% MeOH in EtOAc plus 1% TEA) afforded 0.481 g of compound 196 and 1.315 g of compound 197.

Compound 196: LCMS: MS (ESI) m/z 1073.25 [M+Na] ⁺

Compound 197: LCMS: MS (ESI) m/z 995.23 [MH] ^-

Compound 188

To a solution of TEA salt compound 197 (1.315 g, 1.197 mmol) in MeCN (20 ml) was added 2-nitrobenzyl bromide (0.388 g, 1.796 mmol) at ambient temperature. The resulting solution was stirred at ambient temperature while monitoring the reaction by LCMS. Upon completion, the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography ( _SiO2 100 g, 0%-5% MeOH in EtOAc) to give 0.90 g of compound 188.

LCMS: MS(ESI) m/z 1154.29[M+Na] ⁺

Compound 198

To a solution of compound 188 (1.35 g, 1.192 mmol) in toluene (540 ml) at mild reflux (110-112 °C internal temperature) was added the second generation Hoveyda-Grubbs catalyst (0.187 g, 0.298 mmol) and quinone (0.322 g) , 2.981 mmol) in toluene (20 mL). The resulting solution was stirred at 110-112°C while monitoring the reaction by LCMS. After 4 hours, additional catalyst (0.10 g, 0.16 mmol) was added and stirring at reflux was continued. Upon completion, the reaction mixture was cooled to ambient temperature and treated with DMSO (2.54 ml, 35.8 mmol). After stirring for 3 hours, the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography ( _SiO2 50 g, 0%-15% MeOH in EtOAc) to give a mixture of compound 198 and compound 199. The mixture was dissolved in dichloromethane (2 mL) and treated with water (0.021 ml, 1.2 mmol) and 6% dichloroacetic acid (0.098 ml, 1.2 mmol) in dichloromethane (2 mL). After 10 minutes, the reaction was quenched with pyridine (1 ml) and concentrated in vacuo. The residue was dissolved in dichloromethane (50 mL), washed twice with 30% aqueous NaCl (15 mL each), and dried over _MgSO4 . The crude product was purified by silica gel column chromatography ( _SiO2 50 g, 5%-10% MeOH in DCM) to give 0.25 g of compound 198.

Compound 199: LCMS: MS (ESI) m/z 1104.31 [M+H] ⁺

Compound 198: LCMS: MS (ESI) m/z 802.16 [M+H] ⁺

Compound 190

Compound 198 (0.233 g, 0.291 mmol) and 3-(bis(diisopropylamino)phosphinooxy)propionitrile (0.111 ml, 0.349 mmol) were dissolved in MeCN (10 mL) and concentrated in vacuo. The same operation is repeated two more times. The resulting mixture was dissolved in dichloromethane (2.3 ml), cooled to 0-5 °C, and treated with diisopropylammonium tetrazolium (0.025 g, 0.145 mmol). The reaction mixture was warmed to ambient temperature overnight and then diluted with acetonitrile (2.3 ml). The resulting solution was added via a syringe pump to a solution of pyridine trifluoroacetate (0.168 g, 0.872 mmol) in MeCN (18.6 ml) over 7 hours. Then 3-(bis(diisopropylamino)phosphinooxy)propionitrile (40 mg, 0.133 mmol) in MeCN (2 mL) was added over 2 hours. After stirring for 1 hour, (E)-N,N-dimethyl-N'-(3-thione-3H-1,2,4-dithiazol-5-yl)formamidine (0.119 g, 0.581 g) was added mmol), the reaction solution was stirred until the sulfidation was complete (monitored by LCMS). Upon completion, the mixture was concentrated in vacuo, dissolved in MTBE (5ml) and treated with saturated aqueous _NaHCO3 (3.50ml) and water (1.2ml). The layers were separated and the aqueous layer was extracted with a MTBE/EtOAc mixture (5/3 ml). The combined organic layers were washed twice with 30% aqueous NaCl (2.3 ml), dried over _MgSO4 , filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography ( _SiO2 25g, 0%-20% MeOH in EtOAc) to give 119 mg of compound 190 (58 mg of the fast eluting isomer and 61 mg of the slow eluting isomer).

Compound 191 (SpRp isomer)

To a solution of the fast-eluting isomer of compound 190 (58 mg, 0.062 mmol) in dichloromethane (1.8 mL) was added tert-butylamine (1.2 mL, 11 mmol). The resulting solution was stirred at ambient temperature until all starting material was consumed (monitored by LCMS). Upon completion, the reaction mixture was concentrated in vacuo, azeotroped twice with acetonitrile, and dissolved in acetonitrile (1.7 ml). To the resulting solution was added 1-(bromomethyl)-2-nitrobenzene (40.3 mg, 0.187 mmol). The reaction mixture was stirred at ambient temperature while monitoring the reaction by LCMS. After complete alkylation, the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography ( _SiO2 10 g, 0%-5% MeOH in EtOAc) to afford 19 mg of compound 191 (SpRp isomer).

¹ H NMR (400MHz, chloroform-d) δ=8.42(s,1H), 8.27(s,1H), 8.13(dd, J=1.2, 8.2Hz,1H), 8.05(s,1H), 7.98(d ,J=7.8Hz,1H),7.69-7.67(m,1H),7.61-7.34(m,5H),6.33(d,J=17.2Hz,1H),6.19(d,J=19.9Hz,1H) ,6.09-5.93(m,1H),5.82-5.66(m,2H),5.67(dd,J＝3.5,50.8Hz,1H),5.35-5.32(m,2H),4.62-4.29(m,11H) , 3.35-3.28(m, 1H), 3.18-3.14(m, 2H), 3.01-2.91(m, 1H), 2.84-2.78(m, 1H), 2.60-2.49(m, 3H).

Compound 192 (RpRp isomer)

The slow eluting isomer of compound 190 (60 mg) was processed by the same reaction sequence as described for compound 191 to give 14 mg of compound 192 and 7 mg of compound 191.

¹ H NMR (400 MHz, chloroform-d) δ=8.18 (s, 2H), 8.10 (s, 2H), 8.10 (dd, J=1.0, 8.0 Hz, 2H), 7.66-7.57 (m, 4H), 7.53 -7.46(m,2H),6.31(d,J=17.6Hz,2H),6.19-6.05(m,2H),5.84(dd,J=4.3,50.8Hz,2H),5.39-5.30(m,2H) ),4.58-4.46(m,6H),4.45-4.39(m,2H),4.19(ddd,J=3.1,6.3,10.2Hz,2H),3.32(td,J=3.5,15.6Hz,2H), 3.06 (td, J=6.3, 15.2 Hz, 2H), 2.67-2.57 (m, 4H).

Based on the synthetic methodology, NMR data (symmetric), HPLC retention time (slowest eluting isomer) and biological activity of compound 34 derived from compound 192, Applicants believe that compound 192 has a RR phosphorous stereochemistry. This stereochemical assignment was confirmed by X-ray crystallography.

Compound 33

To a solution of compound 192 (26 mg, 0.026 mmol) in 1,4-dioxane (0.52 ml) was added thiophenol (0.26 ml, 2.5 mmol) followed by TEA (0.26 ml, 1.9 mmol). The resulting mixture was stirred at ambient temperature until deprotection was complete (monitored by LCMS). Upon completion, water (2 mL) was added. The resulting mixture was extracted three times with toluene (2 mL each). The aqueous layer was concentrated in vacuo at 40-50°C and dissolved in water (2 mL). The resulting mixture was extracted four times with an EtOAc/MTBE mixture (1/1 mL each). The aqueous layer was concentrated in vacuo to give compound 33 as the bisTEA salt.

¹ H NMR (400MHz, methanol-d ₄ )δ=9.06(s,1H), 8.73(s,1H), 8.56(s,1H), 8.47(s,1H), 6.43(d,J=14.4Hz, 1H), 6.38(d, J＝15.2Hz, 1H), 6.27(dd, J＝3.5, 51.1Hz, 1H), 5.63(dd, J＝2.7, 51.5Hz, 1H), 5.30-5.14(m, 2H ),5.06-4.91(m,2H),4.58(br d,J=12.5Hz,1H),4.52-4.39(m,3H),4.07(dd,J=4.7,11.7Hz,1H),3.98(dd , J=5.1, 12.5Hz, 1H), 3.43-3.34(m, 2H), 3.22(q, J=7.2Hz, 12H), 2.98-2.86(m, 2H), 2.79-2.45(m, 4H), 1.32(t,J=7.4Hz,18H)

Compound 34

Compound 34 was prepared by the same reaction sequence as described for compound 33 using compound 192 as starting material.

¹ H NMR (400 MHz, methanol-d ₄ ) δ=8.77 (s, 2H), 8.49 (s, 2H), 6.39 (d, J=15.2 Hz, 2H), 5.71 (dd, J=3.1, 51.2 Hz, 2H),5.24-5.13(m,2H),5.02(dtd,J=3.1,9.0,25.4Hz,2H),4.54(br d,J=12.1Hz,2H),4.42(br d,J=9.8Hz ,2H),3.99(dd,J＝5.9,12.1Hz,2H),3.20(q,J＝7.3Hz,12H),2.90(ddd,J＝3.5,10.2,14.1Hz,2H),2.81-2.70( m, 2H), 2.55-2.43 (m, 2H), 1.30 (t, J=7.4Hz, 18H).

Example 22--Synthesis of Compound 35 and Compound 36

Compound 201

To the disodium salt of compound 200 (0.10 g, 0.126 mmol) was added 2-nitrobenzyl bromide (0.068 g, 0.316 mmol) and MeCN (2.0 ml). The resulting mixture was stirred at ambient temperature while monitoring the reaction by LCMS. Upon completion, the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography ( _SiO2 10 g, 0%-5% MeOH in DCM) to give 115 mg of compound 201.

¹ H NMR (400MHz, chloroform-d) δ=8.13 (dd, J=0.8, 8.2Hz, 1H), 8.06 (s, 1H), 8.03 (dd, J=1.0, 8.0Hz, 1H), 7.80 (br s,1H),7.75(s,1H),7.70-7.65(m,1H),7.62-7.54(m,2H),7.53-7.47(m,2H),7.45-7.39(m,1H),7.34- 7.27(m,1H),6.23(br d,J=16.8Hz,1H),6.14(br d,J=18.8Hz,1H),5.95(br s,2H),5.77(td,J=5.1,15.2 Hz,2H),5.71(td,J＝5.1,15.6Hz,1H),5.63-5.55(m,1H),5.47(br d,J＝11.7Hz,1H),4.58-4.27(m,11H), 4.18-4.10(m,1H),4.05-3.71(m,2H)

Compound 202

To a solution of compound 201 (77 mg, 0.076 mmol) in 2,2,2-trifluoroethanol (2 ml) was added O-(2,4-dinitrophenyl)hydroxylamine (36.5 mg, 0.183 mmol) in one portion and bis[rhodium(α,α,α′,α′-tetramethyl-1,3-benzenedipropionic acid)] (5.74 mg, 0.015 mmol). The mixture was stirred at ambient temperature overnight, diluted with DCM (10 mL), and treated with saturated aqueous NaHCO ₃ (10 mL). The layers were separated and the aqueous layer was extracted twice with DCM (10 mL each). The combined organic layers were dried over _MgSO4 and purified by silica gel column chromatography ( _SiO2 10 g, 0%-15% MeOH in EtOAc) to give 3.3 mg of compound 202.

LCMS: MS m/z 1032.07[M+H] ⁺

Compound 35 and Compound 36

To a solution of compound 202 (3.3 mg, 3.2 μmol) in 1,4-dioxane (0.40 ml) was added thiophenol (0.2 ml, 1.9 mmol) and TEA (0.2 ml, 1.4 mmol). The resulting mixture was stirred at ambient temperature while monitoring the reaction by LCMS. Upon completion, the reaction mixture was treated with water (2 mL) and extracted three times with toluene (2 mL each). The aqueous layer was concentrated in vacuo at 40-50°C and dissolved in water (1.5 ml). The resulting solid was filtered off and rinsed with water (0.5 mL). The combined filtrates were subjected to HPLC separation under the following conditions to give compound 35 (retention time: 8.4 minutes) and compound 36 (retention time: 8.9 minutes)

Compound 35

LCMS: MS m/z 762.08[M+H] ⁺

Compound 36

LCMS: MS m/z 762.22[M+H] ⁺

Compound 35/Compound 36 preparative HPLC conditions:

Example 23--Synthesis of Compound 38 and Compound 39

FIG. 13 shows synthetic examples of compound 38 and compound 39.

step 1

To a solution of compound 203 (5.82 g, 9.314 mmol) and allyl alcohol (0.95 ml, 14 mmol) in THF (50 mL) at 0 °C was added DIAD (2.4 ml, 11.6 mmol) and triphenylphosphine (2.93 g, 11.2 mmol) in toluene (25 ml) while keeping the internal temperature below 10°C. The resulting solution was warmed to ambient temperature and stirred while monitoring the reaction by LCMS. Upon completion (5 hours), the mixture was concentrated in vacuo and purified by silica gel column chromatography (100 g, 20%-60% EtOAc in n-heptane) to afford 4.56 g of compound 204 as a pink oil.

Compound 204: ¹ H NMR (400 MHz, chloroform-d) δ=8.55 (s, 1H), 8.24 (s, 1H), 7.46 (d, J=8.2 Hz, 2H), 7.31-7.23 (m, 1H), 7.20-7.10(m, 2H), 6.02(d, J=5.1Hz, 1H), 6.07-5.95(m, 1H), 5.21(dd, J=1.6, 17.2Hz, 1H), 5.05(dd, J= 1.4,10.4Hz,1H),5.00(d,J=5.5Hz,2H),4.82(t,J=4.9Hz,1H),4.22-4.16(m,1H),4.05-4.00(m,2H), 3.82(dd,J=2.5,11.5Hz,1H),0.95(s,9H),0.81(s,9H),0.14(s,3H),0.13(s,3H),0.00(s,3H),- 0.23(s, 3H).

Step 2

To compound 204 (4.56 g, 6.858 mmol) were added acetic acid (80 ml) and water (20 ml). The resulting mixture was stirred at ambient temperature overnight and at 35°C for 1 day. The resulting mixture was concentrated in vacuo and purified by silica gel column chromatography ( _SiO2 , 50 g, 33%-60% EtOAc in n-heptane) to afford 3.22 g of compound 205 as a white foamy solid.

Compound 205: ¹ H NMR (400 MHz, chloroform-d) δ=8.56(s,1H), 7.88(s,1H), 7.51-7.47(m,2H), 7.32-7.27(m,1H), 7.20-7.16 (m, 2H), 6.06-5.95 (m, 1H), 5.72 (d, J=7.8Hz, 1H), 5.26 (dd, J=5.5, 7.8Hz, 1H), 5.20 (dd, J=1.6, 17.2 Hz, 1H), 5.06(dd, J=1.6, 10.2Hz, 1H), 5.03-4.98(m, 2H), 4.23(d, J=5.5Hz, 1H), 4.14(s, 1H), 3.92(dd , J=1.6, 12.9Hz, 1H), 3.68 (br d, J=12.9Hz, 1H), 0.75 (s, 9H), -0.14 (s, 3H), -0.62 (s, 3H).

Step 3

To a solution of compound 205 (3.22 g, 5.847 mmol) in THF (120 mL) at ambient temperature was added water (30 ml) and triphenylphosphine (2.454 g, 9.355 mmol). The resulting mixture was stirred at ambient temperature while monitoring the reaction by LCMS. After completion (18 hours), the reaction mixture was concentrated in vacuo and azeotroped three times with MeCN. The residue was purified by silica gel column chromatography ( _SiO2 (NH) 55g, 20%-100% EtOAc in n-heptane) to give 4.82g of compound 206 (63% purity assumed). The product was used in the next step without further purification.

Compound 206: ¹ H NMR (400 MHz, chloroform-d) δ=8.56 (s, 1H), 7.92 (s, 1H), 7.54 (qd, J=1.6, 7.6 Hz, 2H), 7.31-7.27 (m, 1H) ),7.19-7.15(m,2H),6.07-5.94(m,1H),5.84(d,J＝6.6Hz,1H),5.50(br d,J＝10.6Hz,1H),5.20(dd,J =1.6,17.2Hz,1H),5.05(dd,J=1.4,10.4Hz,1H),5.02-4.98(m,2H),4.95-4.91(m,1H),4.16-4.08(m,2H), 3.96(dd,J=1.6,12.9Hz,1H),3.73(dd,J=2.0,5.5Hz,1H),3.73-3.67(m,1H),0.78(s,9H),-0.20(s,3H ), -0.46(s, 3H).

Step 4

To a solution of compound 206 (4.82 g, 63% pure, 5.72 mmol) in pyridine (39.0 ml) at ambient temperature was added TEA (1.3 ml, 8.6 mmol) and trityl-Cl (1.753 g, 6.289 mmol). After complete reaction (monitored by LC/MS), saturated _NaHCO3 solution (60 mL) was added. The resulting mixture was extracted three times with a MTBE/EtOAc mixture (1/1, 70 mL each). The combined organic layers were washed with 30% aqueous NaCl (30 mL), dried over _MgSO4 , filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography ( _SiO2 50 g, pretreated with 1% TEA in EtOAc/n-heptane, 20%-100% EtOAc in n-heptane plus 1% TEA) to give 1.484 g Compound 207 as a white solid.

Compound 207: ¹ H NMR (400 MHz, chloroform-d) δ=8.49(s,1H), 8.10(s,1H), 7.50-7.46(m,8H), 7.31-7.22(m,7H), 7.20-7.14 (m, 5H), 6.11 (d, J=6.3Hz, 1H), 6.07-5.94 (m, 1H), 5.20 (dd, J=1.2, 17.2Hz, 1H), 5.06 (dd, J=1.4, 10.4 Hz,1H),5.03-4.98(m,2H),4.84(dd,J=3.1,10.6Hz,1H),4.52(t,J=5.7Hz,1H),3.78(d,J=1.6Hz,1H) ), 3.64-3.57(m, 1H), 3.26-3.16(m, 3H), 0.77(s, 9H), -0.18(s, 3H), -0.63(s, 3H).

Step 5

To a solution of compound 207 (0.50 g, 0.652 mmol) in MeCN (6 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (0.393 g, 1.304 g) at ambient temperature mmol) and pyridinium trifluoroacetate (0.113 g, 0.587 mmol). The resulting solution was stirred at ambient temperature while being monitored by LCMS. After completion (1 hour), the resulting solution was treated with hydrogen sulfide gas (bubbling) for 1 minute, then pyridinium trifluoroacetate (0.252 g, 1.304 mmol). The resulting solution was stirred at ambient temperature while being monitored by LCMS. Upon completion (1 hour), the resulting mixture was diluted with MTBE (60 mL). The resulting solution was washed twice with water (15 mL each) and with 30% aqueous NaCl (15 mL), dried _{over Na2SO4} , filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography ( _SiO2 25g, 25%-40% EtOAc in n-heptane) to give 0.411 g of compound 208 as a white foamy solid (1:1P diastereomeric mixture) .

Compound 208 (1:1P diastereomeric mixture) ¹ H NMR (400 MHz, chloroform-d) δ=8.49(s,1H), 8.49(s,1H), 8.11(s,1H), 8.06(s ,1H),7.54-7.47(m,4H),7.46-7.38(m,14H),7.32-7.27(m,2H),7.23-7.04(m,20H),6.11-5.99(m,2H),5.98 (d, J=2.0Hz, 1H), 5.91 (d, J=1.6Hz, 1H), 5.27 (dd, J=1.6, 6.3Hz, 1H), 5.23 (dd, J=1.6, 6.3Hz, 1H) ,5.12(dd,J＝1.6,6.3Hz,1H),5.09(dd,J＝1.2,6.3Hz,1H),5.02(br d,J＝5.5Hz,4H),4.57-4.45(m,2H) ,4.43-4.34(m,1H),4.32-4.04(m,8H),3.23-3.19(m,1H),3.10(br s,2H),3.06-2.99(m,1H),2.96-2.89(m ,2H),2.73(dt,J＝1.4,6.4Hz,2H),2.65(t,J＝6.3Hz,2H),0.85(s,9H),0.83(s,9H),0.01(s,3H) ,-0.09(s,6H),-0.11(s,3H).

Step 6

To a solution of compound 208 (0.411 g, 0.457 mmol) and compound 206 (0.293 g, 0.502 mmol) in acetonitrile (5 ml) at ambient temperature was added DIEA (0.16 ml, 0.91 mmol) and _CCl4 (0.18 ml, 1.8 mmol). The reaction mixture was stirred at ambient temperature while being monitored by LCMS. After completion (1 h), the resulting mixture was concentrated in vacuo and purified by silica gel column chromatography ( _SiO2 25 g, 33%-100% EtOAc in n-heptane) to give 0.461 g of compound 210 (1 ) as a white foamy solid : 1P diastereomeric mixture).

Compound 210: ¹ H NMR (400 MHz, chloroform-d) δ=8.52(s,1H), 8.51(s,1H), 8.49(s,1H), 8.49(s,1H), 8.24(s,1H), 8.19(s,1H),8.13(s,1H),8.01(s,1H),7.55-7.37(m,20H),7.33-6.99(m,30H),6.09-5.97(m,4H),5.96( d,J＝2.7Hz,1H),5.87(d,J＝1.2Hz,1H),5.82(d,J＝4.7Hz,1H),5.80(d,J＝3.9Hz,1H),5.28-5.17( m, 4H), 5.11-5.04 (m, 4H), 5.03-4.96 (m, 8H), 4.75 (t, J=5.1Hz, 1H), 4.70 (t, J=4.5Hz, 1H), 4.46-4.33 (m,3H),4.31-4.18(m,5H),4.12-3.96(m,8H),3.89(br d,J=11.7Hz,1H),3.78-3.67(m,3H),3.57(dd, J=2.9, 4.1Hz, 1H), 3.56-3.44(m, 2H), 3.08-3.01(m, 2H), 2.99-2.93(m, 1H), 2.91(d, J=8.6Hz, 1H), 2.77 -2.73(m,1H),2.72-2.53(m,4H),0.84(s,9H),0.84(s,9H),0.84(s,9H),0.82(s,9H),0.00(s,3H ),-0.05(s,6H),-0.08(s,3H),-0.12(s,3H),-0.15(s,3H),-0.18(s,3H),-0.18(s,3H).

Step 7

To a solution of compound 210 (0.461 g, 0.324 mmol) in MeCN (5.5 ml) at ambient temperature was added 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (0.195 g, 0.648 mmol) and pyridinium trifluoroacetate (0.050 g, 0.259 mmol). The resulting solution was stirred at ambient temperature while being monitored by LCMS. Upon completion (40 minutes), the resulting solution was bubbled with hydrogen sulfide gas for 1 minute and then treated with pyridinium trifluoroacetate (0.138 g, 0.713 mmol). Upon completion (monitored by LCMS), the resulting solution was diluted with MTBE (30 mL), washed twice with water (10 mL each), and 30% aqueous NaCl (10 mL), dried _{over Na2SO4} , filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography ( _SiO2 25g, 33%-60% EtOAc in n-heptane) to give 0.329g of compound 211 as a white foamy solid.

Compound 211: LC/MS (ESI) m/z 1555.48 [M+H] ⁺ .

Step 8

To a solution of compound 211 (0.329 g, 0.211 mmol) in dichloromethane (7.2 ml) was added water (0.032 ml, 1.8 mmol) and dichloroacetic acid (0.29 ml, 3.6 mmol) at ambient temperature. The resulting mixture was stirred at ambient temperature while being monitored by LCMS. After completion (30 min), the reaction mixture was treated with saturated _NaHCO3 solution (25 mL). The resulting mixture was extracted twice with DCM (30 mL each). The combined organic layers were washed with 30% aqueous NaCl (20 mL), dried over _MgSO4 , filtered, and concentrated in vacuo. The crude product compound 212 (0.278 g theoretical yield) was used in the next step without further purification.

Compound 212: LC/MS (ESI) m/z 1313.40 [M+H] ⁺ .

Step 9

To a solution of compound 212 (0.278 g, 0.212 mmol) in MeCN (55.6 ml) was added triethylamine (1.0 ml, 7.2 mmol) and _CCl4 (1.0 ml, 10 mmol) at ambient temperature. The resulting solution was stirred at ambient temperature while being monitored by LCMS. Upon completion (30 minutes), the reaction mixture was concentrated in vacuo to about 20 mL and diluted with MTBE (30 mL). The resulting mixture was washed with water (10 mL) and twice with 30% aqueous NaCl (10 mL each), dried over _MgSO4 , filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography ( _SiO2 25g, 20%-75% EtOAc in n-heptane) to give 71 mg of compound 213 (SpRp isomer), 43 mg of compound 214 (RpRp or SpSp isomer, Higher rf on TLC( _SiO2 ) and 24 mg of compound 215 (RpRp or SpSp isomer, lower rf on TLC( _SiO2 )).

Compound 213 (SpRp isomer) ¹ H NMR (400MHz, chloroform-d)δ=8.58(s,1H), 8.44(s,1H), 8.42(s,1H), 8.12(s,1H), 7.46( dd,J=7.2,15.4Hz,4H),7.36-7.27(m,2H),7.18(q,J=7.6Hz,4H),6.09(s,1H),5.98(s,1H),6.06-5.95 (m,2H),5.25(d,J=11.3Hz,1H),5.21(dd,J=1.4,10.4Hz,1H),5.12-5.03(m,3H),5.00-4.92(m,3H), 4.87(d,J＝4.7Hz,1H),4.60(brd,J＝11.7Hz,1H),4.55(d,J＝3.9Hz,1H),4.44(brd,J＝11.3Hz,1H),4.39 -4.32(m,2H),4.26-4.08(m,8H),3.75(dd,J=11.3,16.0Hz,1H),3.49(dd,J=8.2,10.6Hz,1H),2.82-2.72(m ,3H),2.61(td,J＝5.9,17.2Hz,1H),0.99(s,9H),0.96(s,9H),0.32(s,3H),0.23(s,6H),0.22(s, 3H).

Compound 214 (RpRp or SpSp isomer, higher rf isomer on TLC (SiO ₂ )): ¹ H NMR (400 MHz, chloroform-d) δ = 8.60 (s, 2H), 8.18 (s, 2H) ),7.50(d,J=7.0Hz,4H),7.37-7.31(m,2H),7.25-7.21(m,4H),6.05-5.93(m,4H),5.22(dd,J=1.6,17.2 Hz,2H),5.09(dd,J=1.0,10.4Hz,2H),5.00-4.83(m,6H),4.61(dd,J=1.2,11.7Hz,2H),4.25(dq,J=4.3, 10.8Hz, 2H), 4.09 (brdd, J=3.7, 10.4Hz, 2H), 4.04 (td, J=5.5, 10.9Hz, 2H), 3.99-3.88 (m, 4H), 3.70 (dd, J=11.9 ,15.0Hz,2H),2.71(td,J＝5.5,17.1Hz,2H),2.49-2.37(m,2H),0.98(s,18H),0.25(s,6H),0.24(s,6H) .

Compound 215 (RpRp or SpSp isomer, lower rf isomer on TLC ( _SiO2 )): ¹ H NMR (400 MHz, chloroform-d) δ = 8.54 (s, 2H), 8.06 (s, 2H) ), 7.47-7.44(m, 4H), 7.32-7.27(m, 2H), 7.20-7.15(m, 4H), 6.06-5.95(m, 2H), 5.92(d, J=3.5Hz, 2H), 5.22(dd,J＝1.2,17.2Hz,2H),5.07(dd,J＝1.4,10.4Hz,2H),5.04-4.94(m,6H),4.62-4.52(m,2H),4.46-4.40( m,2H),4.40-4.22(m,8H),3.81(dd,J=6.6,12.1Hz,2H),2.89-2.71(m,4H),0.88(s,18H),0.08(s,6H) ,-0.07(s,6H).

Step 10

To a refluxing solution of compound 213 (71 mg, 0.054 mmol) in toluene (28.4 ml) was added the second generation Hoveyda-Grubbs catalyst (17.0 mg, 0.027 mmol) and p-benzoquinone (11.70 mg, 0.108 mmol) in toluene (8 mL). ) in the solution. The mixture was heated to reflux and the progress of the reaction was monitored by LC/MS. After 3 hours, additional catalyst (8.5 mg, 0.0135 mmol) in toluene (2.5 mL) was added and the reaction continued for an additional 2.5 hours. After cooling, the mixture was concentrated in vacuo and purified by silica gel column chromatography ( _SiO2 10 g, 33%-66% ethyl acetate in n-heptane) to give 17 mg of compound 216 (trans/cis) as a brown dry foam = 5/1).

Compound 216: ¹ H NMR (trans isomer only, 400 MHz, chloroform-d) δ=8.37(s,1H), 8.34(s,1H), 8.24(s,1H), 8.20(s,1H), 7.41(d,J=7.1Hz,2H),7.36-7.27(m,3H),7.24-7.13(m,5H),5.97(d,J=5.9Hz,2H),5.75(td,J=5.5, 15.2Hz, 1H), 5.69(td, J=5.5, 15.6Hz, 1H), 5.07(d, J=3.9Hz, 1H), 5.03(dd, J=5.1, 15.2Hz, 1H), 4.94(t, J=5.5Hz, 2H), 4.85 (dd, J=5.1, 15.2Hz, 1H), 4.74 (d, J=3.9Hz, 1H), 4.67 (br d, J=12.1Hz, 1H), 4.45 (br d, J＝10.2Hz, 1H), 4.42-4.34(m, 2H), 4.28-4.19(m, 2H), 4.19-4.06(m, 4H), 3.96-3.84(m, 1H), 3.82-3.68( m,1H),3.56(br dd,J=12.1,14.5Hz,1H),3.33(dd,J=9.0,10.9Hz,1H),2.83-2.78(m,2H),2.72(td,J=5.6 ,16.6Hz,1H),2.39(td,J=6.3,17.2Hz,1H),1.00(s,18H),0.42(s,3H),0.40(s,3H),0.34(s,3H),0.30 (s, 3H).

Compound 217

Compound 214 obtained from step 9 was treated by step 10 alone to give compound 217 (5/1 mixture of trans/cis isomers).

¹ H NMR (trans isomer only, 400 MHz, chloroform-d) δ=8.47(s,2H), 8.14(s,2H), 7.43-7.38(m,4H), 7.36-7.29(m,2H) ,7.22(t,J＝7.0Hz,4H),5.83(s,2H),5.77(t,J＝3.3Hz,2H),5.17(d,J＝3.5Hz,2H),5.11-5.04(m, 2H), 4.70 (br dd, J=3.5, 16.4Hz, 2H), 4.62 (br d, J=12.1Hz, 2H), 4.16-4.08 (m, 6H), 3.93 (br dd, J=3.7, 11.9 Hz, 2H), 3.74-3.64 (m, 2H), 3.49 (t, J=12.9Hz, 2H), 2.42 (td, J=5.9, 18.4Hz, 2H), 2.13 (ddd, J=5.5, 7.8, 16.8Hz, 2H), 1.00(s, 18H), 0.40(s, 6H), 0.34(s, 6H).

Compound 218

Compound 215 obtained from step 9 was treated by step 10 alone to give compound 218.

¹ H NMR (400MHz, chloroform-d) δ=8.14(s, 2H), 8.12(s, 2H), 7.49-7.43(m, 2H), 7.36-7.27(m, 4H), 7.19-7.12(m, 4H), 5.96(s, 2H), 5.67-5.54(m, 2H), 4.95-4.82(m, 4H), 4.69(d, J＝4.3Hz, 2H), 4.60-4.09(m, 12H), 3.63 (dd, J=8.8, 16.2Hz, 2H), 2.93(td, J=5.9, 17.2Hz, 2H), 2.82-2.72(m, 2H), 0.99(s, 18H), 0.30(s, 6H), 0.27(s, 6H).

Step 11

To compound 216 (trans/cis=5/1, 17 mg, 0.013 mmol) was added methylamine solution (2 ml) (33% in EtOH) at ambient temperature. The resulting solution was stirred at ambient temperature while being monitored by LCMS. After completion (1 hour), the reaction mixture was concentrated in vacuo. To the residue was added pyridine (0.9 ml), TEA (0.45 ml) and triethylamine trihydrofluoride (0.36 ml, 2.2 mmol). The resulting mixture was stirred at 50-60°C for 4 hours and cooled to ambient temperature. After complete TBS deprotection (monitored by LCMS), the reaction mixture was treated with methoxytrimethylsilane (1.5 ml, 12 mmol) and stirred for 1 hour. Water (3 mL) was added and the resulting mixture was extracted twice with toluene (3 mL each) and twice with EtOAc (2 mL each). The aqueous layer was filtered through a syringe filter and the filtrate was subjected to preparative HPLC to yield 5.3 mg of compound 38 and 1.1 mg of compound 220.

Compound 38 (SpRp, trans): ¹ H NMR (400 MHz, methanol-d ₄ ) δ = 9.27 (br s, 1H), 8.47 (br s, 1H), 8.21 (br s, 1H), 8.09 (br s ,1H),6.19-5.96(m,2H),5.94-5.71(m,2H),5.13-4.68(m,2H),4.55-4.39(m,2H),4.38-4.23(m,1H),4.20 -3.91(m, 5H), 3.75-3.50(m, 4H).

Compound 220 (SpRp, cis): ¹ H NMR (400 MHz, methanol-d ₄ ) δ = 9.08 (s, 1H), 8.72 (br s, 1H), 8.55 (s, 1H), 8.24 (s, 1H) ,8.18(s,1H),6.22-6.14(m,2H),6.10(d,J＝1.6Hz,2H),5.01(d,J＝3.9Hz,1H),4.41(br d,J＝9.8Hz ,1H),4.34-4.25(m,3H),4.24-4.18(m,1H),4.15(dd,J=6.1,11.5Hz,1H),4.08-3.99(m,4H),3.71-3.57(m , 3H).

Compound 39 and Compound 222

Compound 217 was treated by step 11 alone to afford compound 39 and compound 222.

Compound 39 (trans isomer): ¹ H NMR (400 MHz, methanol-d ₄ ) δ = 8.83 (br s, 1H), 8.43 (br s, 1H), 8.17 (br s, 1H), 8.11 (br s, 1H) s,1H),6.17-5.95(m,2H),5.93-5.63(m,2H),5.10-4.78(m,2H),4.69-4.53(m,1H),4.52-4.35(m,2H), 4.12-3.92 (m, 5H), 3.76-3.44 (m, 4H).

Compound 222 (cis isomer): ¹ H NMR (400 MHz, methanol-d ₄ ) δ=8.71 (br s, 2H), 8.18 (s, 2H), 6.17-6.06 (m, 4H), 4.37 (br d, J=9.8Hz, 2H), 4.33 (d, J=3.9Hz, 2H), 4.27 (br dd, J=7.0, 13.7Hz, 2H), 4.06 (dd, J=7.0, 11.3Hz, 2H) , 4.19-4.03 (m, 2H), 4.01 (br d, J=9.8Hz, 2H), 3.74 (ddd, J=3.9, 6.4, 10.5Hz, 2H).

Compound 40

Compound 218 was treated by step 11 alone to give compound 40: ¹ H NMR (400 MHz, methanol-d ₄ ) δ = 9.34-8.88 (m, 2H), 8.73 (br s, 1H), 8.34-8.02 (m, 2H) ,6.20-5.96(m,2H),5.89(br s,2H),5.25-4.95(m,2H),4.76-4.64(m,1H),4.47-4.22(m,2H),4.20-4.07(m ,2H),4.07-3.93(m,2H),3.82-3.55(m,3H),3.54-3.38(m,2H)

Example 24 - Synthesis of Compound 30

Compound 2 (1.1 mg, 1.4 μmol) was added to a solution of iodomethyl isopropyl carbonate (13.75 mg, 0.056 mmol) in acetone/water (0.4/0.10 ml) at ambient temperature. The resulting mixture was stirred in the dark at ambient temperature while monitoring the reaction by LCMS. After completion (2 hours), the reaction mixture was diluted with water (0.4 mL) and extracted three times with n-heptane (0.5 mL each). Purification of the crude product in the aqueous layer provided 0.5 mg of compound 30.

¹ H NMR (400MHz, chloroform-d) δ=7.92(s, 2H), 7.72(br s, 2H), 6.22(d, J=17.2Hz, 2H), 6.19-6.03(m, 2H), 5.98( br t,J＝6.3Hz,2H),5.82-5.77(m,2H),5.68(dd,J＝2.3,51.6Hz,1H),5.54(dd,J＝10.9,13.7Hz,2H),5.48( dd,J=10.9,12.9Hz,2H),4.94(quin,J=6.2Hz,2H),4.71-4.63(m,2H),4.63-4.56(m,2H),4.50-4.43(m,2H) , 4.22-4.12 (m, 2H), 4.05-3.90 (m, 2H), 1.33 (d, J=4.3Hz, 6H), 1.32 (d, J=4.3Hz, 6H).

Example 103-HAQ STING agonist activity reporter gene analysis

THP1-Dual ^™ cells (InvivoGen, Cat#thpd-nfis) were used for _EC50 determination. The supplier Invivogen (Insight 201402-1) has identified THP1Dual ^™ cells to carry the HAQ STING genotype. Cells were grown and maintained under conditions recommended by the manufacturer. EC ₅₀ determinations were performed following the induction of the interferon regulatory factor (IRF) pathway as described in the manufacturer's manual. Briefly, cells were seeded and treated with different concentrations of compounds for 20 hours while incubating at 37°C, 5% _CO2 . Cells were resuspended and QUANTI-Luc ^™ solution (Cat.#:rep-qlc1) was added. The resulting light emission was measured by a photometer (Envision, PerkinElmer). The obtained signal was plotted and _EC50 calculated using GraphPad Prism7 software.

_EC50 values are reported in Tables 4-8 below. _EC50 values can be derived from a single assay or an average of multiple assays. In front of each table is the structure for viewing that table.

Table 4

*Indicates that the activity could not be measured within the experimental concentration range.

"N/E" means "Not Evaluated".

Human STING EC50 (μM) was measured using the ammonium salt form of each compound in Table 4. table 5

*Indicates that the activity could not be measured within the experimental concentration range.

**Compound 7 is a 1:1 mixture of Compound 11 and Compound 12.

"N/E" means "Not Available". "N.D." means "not determined".

Human STING EC50 (μM) was measured using the ammonium salt form of each compound listed in Table 5.

Table 6

*Indicates that the activity could not be measured within the experimental concentration range.

"N/E" means "Not Evaluated". "N.D." means "not determined".

Human STING EC50 (μM) was measured using the ammonium salt form of each of the compounds listed in Table 6 except for Compound 26, Compound 31, Compound 33 and Compound 34, all of which were tested as bisTEA salts.

Table 7

*Indicates that the activity could not be measured within the experimental concentration range.

Human STING EC50 (μM) was measured using the ammonium salt form of each compound in Table 7.

Table 8

*Indicates that the activity could not be measured within the experimental concentration range.

Human STING EC50 (μM) was measured using the ammonium salt form of each compound in Table 8.

Example 104 - STING variant-specific reporter gene analysis

Human STING has 4 major variants, including WT, HAQ, REF and AQ variants. For example, REF-STING, also known as R232H, occurs in about 14% of the population. R232H is less responsive to bacterial and metazoan cyclic dinucleotides compared to the wild-type allele. Yi G et al., "Single nucleotide polymorphisms of human STING canaffect innate immune response to cyclic dinucleotides" PLoS One 2013;8:e77846 report details of these 4 major variants and other rare variants. STING variant-specific reporter cell lines were established by using THP1-Dual ^™ KO-STING cells (InvivoGen, Cat#thpd-kostg) and three STING variant protein expression vectors. The expression vector for WT STING is shown in Figure 6. For the other two expression vectors, different STING variant sequences were used in the vectors in which the WT STING was replaced by the appropriate nucleotide sequence.

STING variant expression vectors were prepared for WT-STING, REF-STING and AQ-STING and stably transfected into THP1-Dual ^™ KO-STING cells to prepare for WT-STING, REF-STING and AQ, respectively - STING variant-specific reporter gene analysis for STING. _EC50 values were determined as described above in Example 103 for the HAQ STING agonist activity reporter gene assay. The results are shown in Table 9 below. The DNA sequences for these STING variants are shown in SEQ ID NO: 1 (nucleotide sequence of WT human STING), SEQ ID NO: 2 (nucleotide sequence of REF human STING) and SEQ ID NO: 3 (AQ The nucleotide sequence of human STING).

WT man STING:

atgccccactccagcctgcatccatccatcccgtgtcccaggggtcacggggcccagaaggcagccttggttctgctgagtgcctgcctggtgaccctttgggggctaggagagccaccagagcacactctccggtacctggtgctccacctagcctccctgcagctgggactgctgttaaacggggtctgcagcctggctgaggagctgcgccacatccactccaggtaccggggcagctactggaggactgtgcgggcctgcctgggctgccccctccgccgtggggccctgttgctgctgtccatctatttctactactccctcccaaatgcggtcggcccgcccttcacttggatgcttgccctcctgggcctctcgcaggcactgaacatcctcctgggcctcaagggcctggccccagctgagatctctgcagtgtgtgaaaaagggaatttcaacgtggcccatgggctggcatggtcatattacatcggatatctgcggctgatcctgccagagctccaggcccggattcgaacttacaatcagcattacaacaacctgctacggggtgcagtgagccagcggctgtatattctcctcccattggactgtggggtgcctgataacctgagtatggctgaccccaacattcgcttcctggataaactgccccagcagaccggtgaccgggctggcatcaaggatcgggtttacagcaacagcatctatgagcttctggagaacgggcagcgggcgggcacctgtgtcctggagtacgccacccccttgcagactttgtttgccatgtcacaatacagtcaagctggctttagccgggaggataggcttgagcaggccaaactcttctgccggacacttgaggacatcctggcagatgcccctgagtctcagaacaactgccgcctcattgcctaccaggaacctgcagatgacagcagcttctcgctgtcccaggaggttctccggcacctgc ggcaggaggaaaaggaagaggttactgtgggcagcttgaagacctcagcggtgcccagtacctccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaagcccctccctctccgcacggatttctcttga (SEQ ID NO: 1)

REF PEOPLE STING:

atgccccactccagcctgcatccatccatcccgtgtcccaggggtcacggggcccagaaggcagccttggttctgctgagtgcctgcctggtgaccctttgggggctaggagagccaccagagcacactctccggtacctggtgctccacctagcctccctgcagctgggactgctgttaaacggggtctgcagcctggctgaggagctgcgccacatccactccaggtaccggggcagctactggaggactgtgcgggcctgcctgggctgccccctccgccgtggggccctgttgctgctgtccatctatttctactactccctcccaaatgcggtcggcccgcccttcacttggatgcttgccctcctgggcctctcgcaggcactgaacatcctcctgggcctcaagggcctggccccagctgagatctctgcagtgtgtgaaaaagggaatttcaacgtggcccatgggctggcatggtcatattacatcggatatctgcggctgatcctgccagagctccaggcccggattcgaacttacaatcagcattacaacaacctgctacggggtgcagtgagccagcggctgtatattctcctcccattggactgtggggtgcctgataacctgagtatggctgaccccaacattcgcttcctggataaactgccccagcagaccggtgaccatgctggcatcaaggatcgggtttacagcaacagcatctatgagcttctggagaacgggcagcgggcgggcacctgtgtcctggagtacgccacccccttgcagactttgtttgccatgtcacaatacagtcaagctggctttagccgggaggataggcttgagcaggccaaactcttctgccggacacttgaggacatcctggcagatgcccctgagtctcagaacaactgccgcctcattgcctaccaggaacctgcagatgacagcagcttctcgctgtcccaggaggttctccggcacctgc ggcaggaggaaaaggaagaggttactgtgggcagcttgaagacctcagcggtgcccagtacctccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaagcccctccctctccgcacggatttctcttga (SEQ ID NO: 2)

AQ people STING:

atgccccactccagcctgcatccatccatcccgtgtcccaggggtcacggggcccagaaggcagccttggttctgctgagtgcctgcctggtgaccctttgggggctaggagagccaccagagcacactctccggtacctggtgctccacctagcctccctgcagctgggactgctgttaaacggggtctgcagcctggctgaggagctgcgccacatccactccaggtaccggggcagctactggaggactgtgcgggcctgcctgggctgccccctccgccgtggggccctgttgctgctgtccatctatttctactactccctcccaaatgcggtcggcccgcccttcacttggatgcttgccctcctgggcctctcgcaggcactgaacatcctcctgggcctcaagggcctggccccagctgagatctctgcagtgtgtgaaaaagggaatttcaacgtggcccatgggctggcatggtcatattacatcggatatctgcggctgatcctgccagagctccaggcccggattcgaacttacaatcagcattacaacaacctgctacggggtgcagtgagccagcggctgtatattctcctcccattggactgtggggtgcctgataacctgagtatggctgaccccaacattcgcttcctggataaactgccccagcagaccgctgaccgagctggcatcaaggatcgggtttacagcaacagcatctatgagcttctggagaacgggcagcgggcgggcacctgtgtcctggagtacgccacccccttgcagactttgtttgccatgtcacaatacagtcaagctggctttagccgggaggataggcttgagcaggccaaactcttctgccagacacttgaggacatcctggcagatgcccctgagtctcagaacaactgccgcctcattgcctaccaggaacctgcagatgacagcagcttctcgctgtcccaggaggttctccggcacctgc ggcaggaggaaaaggaagaggttactgtgggcagcttgaagacctcagcggtgcccagtacctccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaagcccctccctctccgcacggatttctcttga (SEQ ID NO: 3)

Example 105 - Reporter gene analysis of mouse STING agonist activity

RAW-Lucia ^™ ISG cells (InvivoGen, Cat#rawl-isg) were used for mouse STING agonist reporter gene analysis. _EC50 values were determined as described in Example 103 above in the HAQ STING agonist activity reporter gene assay. The results are shown in Table 9 below.

Example 106 - Differential Scanning Fluorimetry (DSF) Analysis

DSF analysis was used to measure physical interactions between compounds and recombinant STING protein. The truncated recombinant STING protein (aa155-341) (SEQ ID NO:4) was expressed in E. coli and isolated for analysis as described below. Assay matrices were prepared in 384-well plates in a final volume of 10 μL per well consisting of 1 μM recombinant STING protein (aa155-341) (SEQ ID NO: 4), 100 mM PBS pH 7.4 (supplemented with 100 mM KCl), 5X SYPRO orange dye and 50 μM compound (final DMSO concentration 0-1%). Analysis was performed on a QuantStudio 12K Flex real-time PCR system using a temperature gradient of 25°C to 95°C at a rate of 0.05°C/min, with excitation and emission filters of 470 nm and 586 nm, respectively. According to Applied The thermal melting temperature (Tm) and the thermal melting temperature difference (dTmD) of the unbound and ligand-bound recombinant STING proteins were calculated from the fluorescence derivative curves specified by the protein thermal shift software (algorithm version 1.3.).

In general, compounds with ΔTm values greater than 0 are considered to have physical interactions with the test protein, and ΔTm values positively correlate with the binding affinity of the compound. Here, compound 1a exhibited a ΔTm of 17.6 (Table 9), indicating a physical interaction with the STING protein.

Table 9 In vitro characterization of compound 1a

Example 107--In vitro human PBMC stimulation analysis

Human blood was collected from 5 healthy donors using 10.0 mL BD Vacutainer sodium heparin tubes (cat#367874). Isolation of peripheral blood mononuclear cells (PBMC) was performed using SIGMA ACCUSPIN 50 ml tubes (cat#A2055) and sigma ACCUSPIN System-HISTOPAQUE-1077 (cat#A7054) using the protocol provided by the manufacturer. The PBMC layer was harvested and washed with IX Phosphate Buffered Saline (PBS) as recommended by Sigma. PBMCs were counted and finally suspended at 1 x 10e6/ml in RPMI (corning cat #10-041-CV) supplemented with 10% fetal bovine serum (FBS) (Gibco cat #20140.79). 1 ml of cells (1 x 10e6) were transferred to Falcon 5 mL round bottom polypropylene tubes (cat#352063) and incubated with different concentrations (0 uM, 0.1 uM, 1 uM) in a 5% CO2 incubator at 37°C. , 10 uM) stimulation for 24 hours.

After 24 hours of incubation, the tubes were centrifuged at 1400 rpm for 5 minutes and the supernatant was harvested. The supernatant was stored at -80°C for subsequent IFNβ measurements. IFN[beta] measurements were performed using the Human IFN-[beta] Basic Kit (Meso Scale Diagnosticscat #K151ADA) using the protocol provided by the manufacturer. IFN-β estimation was done by reading the analysis plate of MESO SECTOR Imager2400 and using the MSD Discovery Workbench 4.0 program. IFN[beta] protein was analyzed after 24 hours. The results showed that compound 1a was able to induce IFNβ protein production in primary human PBMCs in a dose-dependent manner.

The results shown in Table 10 reflect the average of measurements performed using five different donors.

Table 10 In vitro human PBMC stimulation analysis

For quantification of IFN[beta] mRNA, total RNA was isolated using the RNeasy mini kit (Qiagen, Germany) according to the manufacturer's protocol. IFN[beta] mRNA was quantified by qPCR analysis. Briefly, total RNA (400 ng to 1000 ng) was converted to cDNA using SuperScript VILO MasterMix (Life Technologies, USA) in a 60 μl reaction volume. The obtained cDNA (10 ng) was subsequently amplified using Applied Biosystems TaqMan expression analysis using RNA specific primers for IFNB1 (Hs01077958_s1) and GAPDH (Hs99999905_m1). qPCR analysis was performed on an Applied Biosystems Quantstudio 12K Flex real-time PCR system with TaqMan Fast AdvancedMaster Mix (Life Technologies, USA), first at 50°C for 2 min, then at 95°C for 2 s, and 40 cycles of 95°C for 1s and 60°C for 20s. Relative gene expression was calculated using the 2-ΔΔCT method after normalization to the reference gene GAPDH. Calculations were performed using Applied Biosystems Quantstudio 12KFlex software v1.2.2. Table 11 summarizes the fold change of IFN[beta] mRNA relative to vehicle treated samples. The results showed that compound 1a was able to induce IFNβ mRNA in primary PBMCs in a dose- and time-dependent manner. Table 11 shows the mean values calculated from five different donors.

Table 11--Ex vivo Human PBMC 3-Hour and 24-Hour Stimulation Analysis (mRNA)

Example 108--Anticancer effect of compound 1a on CT26 dual tumor model

The anticancer activity of Compound 1a was tested in the CT26 dual tumor model, which is a mouse colon cancer model. 5-6 week old female Balb/cJ mice (Jackson Labs, Bar Harbor, Maine) were implanted with CT26 tumor cells subcutaneously on both sides of each animal, 10 ⁵ cells per side. For Study A, treatment (1.25 mg/kg, 2.5 mg/kg and 5 mg/kg) was initiated 5 days after tumor implantation, at which point the mean tumors reached approximately 100 ^mm3 . For Study B, treatment (0.6 mg/kg and 10 mg/kg) was initiated 8 days after tumor implantation, at which point the mean tumors reached approximately 120 ^mm3 . Treatment regimens are described in Table 12 and Table 13.

Table 12 Dosing regimen for Study A

*I.T. is intratumoral.

Table 13 Dosing regimen for Study B

*I.T. is intratumoral.

All mice in the study had two subcutaneous CT26 tumors. A "treated tumor" refers to a tumor to which the compound has been directly administered, while an "untreated tumor" refers to a tumor to which the compound has not been directly administered. Tumor volume was tracked throughout the experiment. After initiation of treatment, tumor volumes were measured twice a week. Tumor burden was calculated from caliper measurements by the volume formula for prolate spheroids (L×W ² )/2, where L and W are orthogonal length and width measurements (mm), respectively.

Compound 1a showed potent therapeutic activity in the CT26 dual tumor model (Figures 7 and 8). For treated tumors, a 20% cure rate was detected even at the lowest dose tested in the study (Figure 8, 0.6 mg/kg dose). Meanwhile, at the end of the study, the highest dose (10 mg/kg) cured 100% of the tumor animals. A dose-dependent antitumor effect was also evident for untreated tumors. The highest dose group (10 mg/kg) showed 80% efficacy; all lower doses also showed tumor growth inhibitory activity. Thus, a therapeutic window of 0.6 mg/kg to 10 mg/kg was observed for Compound 1a, with not only local but systemic antitumor activity based on effects at non-injected distal tumor sites. Taken together, these results suggest that topical administration of Compound 1a induces both local and systemic (distal) anticancer activity.

Example 109--Anticancer effect of compound 1a on CT26 liver metastasis model

The anticancer activity of compound 1a was tested in the CT26 liver metastasis model. Anesthetized 5-6 week old female BALB/cJ mice (Jackson Labs, Bar Harbor, Maine) were intraspleen implanted with luciferase-expressing CT26 tumor cells ( ⁵ x 105 cells per mouse). A subsequent ten minute waiting period allowed tumor cells to circulate into the animal's liver. The spleen was then removed, and the animal was sutured and allowed to recover. Three days later, CT26 tumor cells (105 cells per mouse) ^were implanted again, this time subcutaneously (sc) at the right forelimb site, to form a tumor mass for compound administration. Nine days after intrasplenic injection, compound (10 mg/kg) was administered intratumorally into sc tumors in a single dose.

The local anticancer effect of the compound was measured by its effect on sc tumors, while the distal effect of the compound was based on the deleterious effect of the tumor mass growing in the liver of each mouse by comparison with vehicle-treated control mice. The overall survival rate of the treated mice was assessed. Compound 1a showed potent activity against localized sc tumors and therapeutic systemic activity in 9 of 10 treated animals (Figure 9). These results suggest that topical administration of Compound 1a induces local and systemic (distal) anticancer activity, including deep lesions, such as in the liver.

Example 110--Anticancer effect of compound 1a on GL261 brain orthotopic model

The anticancer activity of compound 1a was tested in the GL261 brain orthotopic model. GL261 is a murine glioma cell line. Luciferase-expressing GL261 mouse glioma cells ( ² x 104 cells/mouse) were implanted intracranially into 5-6 week old female B6 albino mice (Jackson Labs, Bar Harbor, Maine). After 3-4 days, ^GL261 cells (106 cells/mouse) were implanted subcutaneously in the right forelimb site to form tumor masses for compound administration. Ten days after intracranial tumor cell implantation, compounds (10 mg/kg) were administered intratumorally into sc tumors in a single dose. The local anticancer effect of the compound was measured by its effect on sc tumors, while the distal effect of the compound was based on the deleterious effect of the tumor mass growing in the brain of each mouse by comparison with vehicle-treated control mice. The overall survival rate of the treated mice was assessed. Compound 1a showed potent activity against localized sc tumors and therapeutic systemic activity in 5 of 8 treated animals (Figure 10). These results suggest that topical administration of Compound 1a induces local and systemic (distal) anticancer activity, including deep lesions, eg, in the brain.

Example 111 - X-ray structure confirmation complexed with WT STING

To further understand the target-binding mechanism of the new compound, the X-ray crystal structure of WTSTING in complex with the compound was determined.

A. Expression and purification of the WT STING C-terminal domain (residues 155-341)

The DNA sequence encoding amino acids 155-341 of the human WT STING protein (SEQ ID NO:4) was cloned into the pET21b vector with a His-TEV-Sumo tag at its N-terminus (SEQ ID NO:5). The sequence of pET21b has been deposited in addgene, available here: addgene.org/vector-database/2550/; the sequence is incorporated herein by reference.

E. coli BL21 (DE3) codon-plus cells were transformed with this plasmid, and expression of the recombinant protein was induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Proteins were purified from soluble fractions of cell lysates by Ni-NTA affinity chromatography. The His-TEV-Sumo tag was removed with sumo protease and separated from untagged WT STING155-341 using another Ni-NTA affinity column. The protein was further purified by anion exchange and size exclusion chromatography and stored in buffer containing 20 mM Tris·HCl (pH 7.5) and 150 mM NaCl (35 mg/ml).

B. Crystallization and structural determination of the WT STING C-terminal domain in complex with compound 1

To co-crystallize WT STING 155-341 with Compound 1, WT STING protein was diluted to 10 mg/ml using storage buffer (20 mM Tris HCl pH 7.5 and 150 mM NaCl) and mixed with Compound 1 (100 mM stock solution in DMSO) Mix in a molar ratio of 1:5. The mixture was incubated at 4°C for 4 hours and centrifuged at 13,000 rpm for 20 minutes before crystallization. Crystallization sieve trays were assembled at 18°C using the hanging drop vapor diffusion method. Crystals were grown by mixing 1 μL of WT STING/Compound 1 solution with an equal volume of well solution containing 100 mM HEPES pH 7.5, 200 mM CaCl2 and 15% (wt/vol) PEG 8000. When the crystals were snap frozen in liquid nitrogen, 20% (wt/vol) PEG 400 was used as a cryoprotectant. Diffraction datasets were acquired with a Pilatus detector at the SSRF BL19U1 beamline and processed with the HKL3000 and SCALEPACK2MTZ program in the CCP4 software suite.

The structure of WT STING 155-341 bound to compound 1 was determined by molecular replacement using the program PHASER (maximum likelihood molecular replacement) with PDB ID 4F9E as the initial search model. The presence of compound 1 between the dimer interfaces of WT STING was confirmed in the Fo-Fc difference map calculated with the model stage. The model was manually constructed and completed with the Coot program and refined with the Refmac5 program in the CCP4 software suite. In space group P212121, the resolution of the final refined structure is reported to be The measured values of its unit cell are a=33.820, b=78.110, c=132.212, α=90.00, β=90.00, γ=90.00. Two copies of WT STING 155-341 were identified in each asymmetric unit bound to one molecule of compound 1 at the dimer interface.

C. Interaction of compound 1 with WT STING observed in the X-ray crystal structure

FIG. 11 shows a photograph of the X-ray crystal structure of human WT STING complexed with Compound 1. FIG. The X-ray crystal structure of human WT STING complexed with compound 1, which was co-crystallized from a sample of compound 1a, was examined. This compound binds at the interface pocket formed by dimers of the WT STING protein. The two faces of the adenine base of this compound form π-π stacking interactions with the guanidine groups of Tyr240 and Arg238, respectively. The trans-olefin linker forms van der Waals interactions with the aliphatic portion of the side chain of Arg238. The fluorine substituent at the C2' position of the ribose group of the compound is nested in a hydrophobic pore defined by Thr263, Pro264 and Tyr163. The negatively charged phosphorothioate group of this compound forms a salt bridge with Arg238 and a hydrogen bond interaction with Ser162 and Thr267, respectively. In addition, the phosphorothioate group also forms electrostatic interactions with the guanidine group of Arg 232. The LID loop region of WT STING, consisting of residues 226 to 243, surrounds two bases and a trans-olefin linker.

Example 112 - Determination of the X-ray Crystal Structure of REF STING Complexed with Compound 1

A. Expression and purification of the REF STING C-terminal domain (residues 155-341, SEQ ID NO: 6)

The DNA sequence encoding amino acids 155-341 of the human REF STING protein (SEQ ID NO:6) was cloned into the pET21b vector with a His-TEV-Sumo tag at its N-terminus (SEQ ID NO:7). The sequence of pET21b has been deposited in addgene, available here: addgene.org/vector-database/2550/ ; the sequence is incorporated herein by reference.

E. coli BL21 (DE3) codon-plus cells were transformed with this plasmid, and expression of the recombinant protein was induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Proteins were purified from soluble fractions of cell lysates by Ni-NTA affinity chromatography. The His-TEV-Sumo tag was removed with sumo protease and separated from tag-free REF STING_155-341 using another Ni-NTA affinity column. The protein was further purified by anion exchange and size exclusion chromatography and stored in buffer containing 20 mM Tris·HCl (pH 7.5) and 150 mM NaCl (24 mg/ml concentration).

B. Crystallization and structural determination of the C-terminal domain of REF STING in complex with compound 1

To co-crystallize REF STING_155-341 with compound 1, REF STING protein was diluted to 10 mg/ml using storage buffer (20 mM Tris HCl pH 7.5 and 150 mM NaCl) and mixed with compound 1 (100 mM stock solution in DMSO) to Molar ratio of 1:5 mixed. The mixture was incubated at 4°C for 4 hours and centrifuged at 13,000 rpm for 20 minutes before crystallization. Crystallization sieve trays were assembled at 18°C using the hanging drop vapor diffusion method. Crystals were grown by mixing 1 μL of REF STING/Compound 1 solution with an equal volume of well solution containing 100 mM HEPES pH 7.5, 200 mM CaCl2 and 15% (wt/vol) PEG 8000. When the crystals were snap frozen in liquid nitrogen, 20% (wt/vol) PEG 400 was used as a cryoprotectant. Diffraction datasets were acquired with a Pilatus detector at the SSRF BL18U1 beamline and processed with the HKL3000 and SCALEPACK2MTZ program in the CCP4 software suite. This structure is shown in FIG. 12 .

The structure of REF STING_155-341 bound to compound 1 was determined by molecular replacement using the program PHASER (maximum likelihood molecular replacement) using the previously determined structure of WT STING 155-341 (described above) as an initial search model. The presence of compound 1 between the dimer interfaces of REF STING was confirmed in the Fo-Fc difference map calculated with the model stage. The model was manually constructed and completed with the Coot program and refined with the Refmac5 program in the CCP4 software suite. In space group P212121, the resolution of the final refined structure is reported to be The measured values of its unit cell are a=33.733, b=77.831, c=131.689, α=90.00, β=90.00, γ=90.00. Two copies of REF STING 155-341 were identified in each asymmetric unit bound to one molecule of compound 1 at the dimer interface.

C. Interaction of compound 1 with REF STING observed in the X-ray crystal structure

Figure 12 shows the X-ray crystal structure of human REF STING in complex with Compound 1, which was co-crystallized from a sample of Compound 1a. The compound binds at the interface pocket formed by dimers of the STING protein. The two faces of the adenine base of this compound form π-π stacking interactions with the guanidine groups of Tyr240 and Arg238, respectively. The trans-olefin linker forms van der Waals interactions with the aliphatic part of the side chain of Arg238, while the guanidine part of the side chain of Arg238 forms a π-π stacking interaction with the imidazole group of the side chain of His232 from the outside. The olefin linker contacts the interacting pair of side chains of Arg238 and His232. The fluorine substituent at the C2' position of the ribose group of the compound is nested in a hydrophobic pore defined by Thr263, Pro264 and Tyr163. The negatively charged phosphorothioate group of this compound forms a salt bridge with Arg238 and a hydrogen bond interaction with Ser162 and Thr267, respectively. The LID loop region of REF STING, consisting of residues 226 to 243, surrounds two bases and a trans-olefin linker.

Example 113 - Comparison

As reported in Corrales et al., "Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity," Cell Reports (2015) 11:1018-1030 (incorporated herein by reference), use of the present disclosure In a head-to-head comparison of compound 1a, the native STING ligand (2'3' cGAMP) and the purported STING agonist ML RR-S2 CDA, the human STING analysis of WT STING, HAQ STING, AQ STING and REF STING was calculated EC50 value. Analyses were performed as described in the above examples. Note that the analytical values reported in Table 14 are limited to analyses performed in head-to-head comparisons and may not reflect the mean values determined in more trials reported in Table 5 or elsewhere.

Table 14

Table 14 also reports the dissociation binding constant (Kd) of human WT STING binding to each of the three test compounds as measured by isothermal titration calorimetry (ITC). ITC is a microcalorimetric technique that measures thermodynamic properties related to intermolecular interactions. Based on these tests, among the compounds tested, compound 1a appeared to form the strongest bond with WT STING.

Material

Recombinant human wild-type STING (aa, 139-379, H232R) protein was produced by expressing in E. coli a construct encoding the cytoplasmic domain of human WT STING comprising amino acids 139-379.

reagent

The sources of reagents used in this study are as follows:

Protein buffer preparation

STING protein was stored in 5% glycerol in PBS (pH 7.5) at -60°C in 90 μL and 100 μL aliquots at concentrations of 3.0 mg/mL and 20 mg/mL, respectively. On the day of analysis, protein aliquots were thawed, diluted to 400 uL, and centrifuged at least 4 times for 10 min at 14,000 x g in an Eppendorf microcentrifuge using an Amicon Ultra centrifugal filter device (10 k MW cutoff, 0.5 mL) Fluid exchange into PBS followed by final dilution to 20 μM to 30 μM (depending on experiment) with 1×PBS. Protein concentration was determined using a Nanodrop 2000 spectrophotometer and a protein extinction coefficient of 22140 (M" ¹ cm" ¹ ).

Sample Preparation

Provide 200 [mu]L of 1 mM stock solutions of compound 1a, 2'3' cGAMP and MLRR-S2CDA through a 1.5 mL microcentrifuge tube. Before each experiment, the samples were diluted to a concentration of 200uM to 500uM (depending on the experiment).

method

Analysis was performed on an Affinity ITC apparatus (TA Instruments no. 609003.901) equipped with an ITC cleaning accessory (TA Instruments no. 601800.901). Pipette approximately 400 μL of STING protein solution (containing 20 μM-30 μM MSTING protein) into a 185 μL calorimeter cell, allowing some acceptable overload. The reference cell contained an equal amount of Milli-Q water. Incubation was performed at 25°C and 20 x 2.5 μL of 100 μM-300 μM compound was injected. The control software ITC RunVer.3.3.0.0 (TAInstruments) was used to obtain a thermogram consisting of multiple raw thermal peaks ([mu]cal/sec) representing the thermal rate at each injection. Analysis software Nano Analyze Ver. 3.70 (TA Instruments) was used for baseline correction, correction for blank or sample dilution heat (at saturation), and integration of heat rate peaks to generate graphical "Q" values. The resulting isotherms were fitted to independent models to derive thermodynamic parameters.

Derive and report Kd and n values (molar ratio at the inflection point of the curve). Optimal conditions for protein and ligand concentrations were derived from preliminary experiments.

result

Thermal rate thermograms and resulting isotherms of the binding of test compounds to recombinant human wild-type STING (aa, 139-379, H232R) were determined. Binding of each compound to STING was endothermic, manifested by a negative heat rate and an exothermic (positive) heat of dilution (observed after the compound reached protein saturation). 2',3' cGAMPs have been shown to produce similar endothermic responses to various STING variants. Compound 1a had the lowest Kd at 0.04 μM, followed by 2’3’ cGAMP with a Kd of 0.07 μM, followed by MLRR-S2CDA with a Kd of 0.40 μM. The n values for all compounds were close to 0.5, indicating that the STING protein exists in a dimer form, with every 2 mol of STING bound to 1 mol of compound.

Example 114 - Identification of Potential Metabolites

Compound 1a was incubated in hepatocytes from CD-1 mice, Sprague Dawley rats, beagle dogs, cynomolgus monkeys and humans to assess the formation of major metabolites.

Material

Cryopreserved pooled hepatocytes were purchased from ThermoFisher Scientific (Waltham, MA), Xenotech, LLC (Kansas City, KS), and In Vitro ADMET Laboratories (Columbia, MD), and suitable media were purchased from In Vitro ADMET Laboratories (Columbia, MD) MD) and Life Technologies (Carlsbad, CA). AOPI staining solution and phosphate buffered saline were obtained from Corning Life Sciences (Tewksbury, MA) and Nexcelom Bioscience (Lawrence, MA), respectively. All chemicals, reagents and solvents used in the analysis were analytical or HPLC grade.

Experimental Design and Procedures

Hepatocyte Incubation

Compound 1a was weighed and dissolved in HPLC-water containing 0.12% formic acid in PBS to reach 1020 mmol/L. The solution was then diluted 2.5 times to 4 mmol/L with Williams'E medium containing 0.1% human serum albumin and 2 mmol/L L-glutamine, and then further diluted 21000 times to make a concentration of 20 μmol/L working stock solution.

Before incubation, cryopreserved hepatocytes were thawed in a 37°C water bath. One tube of cryopreserved hepatocytes was added to each 50 mL cryopreserved hepatocyte recovery medium (UCRM) conical tube obtained from In Vitro ADMET Laboratories (Columbia, Md). Cells were spun at 740 rpm in a Beckman centrifuge (Brea, CA) with a GH 3.8 rotor at room temperature 4°C for 10 minutes. The supernatant was removed and the cells were resuspended in plating medium and counted. After the cells were resuspended in the plate medium, 20 μL of the resuspension was transferred and mixed with 20 μL of LAOPI staining solution. The solution was mixed gently and cells were counted using a Cellometer (Nexcelom, Lawrence, MA). After counting, cells were resuspended at 1 million or 2 million viable cells/mL in Williams'E medium (pH 7.4) containing 2 mmol/L L-glutamine.

The hepatocyte suspension (50 μL/well) was added to a 48-well plate. 50 microliters of working stock solution containing compound 1a (20 μmol/L) was added to start the reaction. The plate was placed in a tissue culture incubator (5% _CO2 /95% air humidified atmosphere and 37°C) and the reaction was stopped with 200 μL of stop solution at 5, 30, 60, 120, 180 and 240 minutes, the The stop solution consisted of 100% methanol/acetonitrile (1/1, v/v) with 2010 ng/mL furosemide and 0.2 μmol/L (R)-propranolol. The mixture was centrifuged and filtered, and the supernatant was collected for analysis. The final concentration of cryopreserved hepatocytes was ¹ x 106 cells/mL. The final incubation concentration of compound 1a was 10 μmol/L.

LC-MS/MS Conditions for Metabolite Identification

The LC-MS/MS system consisted of a Shimadzu HPLC and an AB-SCIEX TripleTOF 5600 hybrid quadrupole with TOF mass spectrometer (Framingham, MA). Shimadzu HPLC (Kyoto, Japan) consists of a communication bus module (CBM-20A), an autosampler (SIL-30AC) with an additional rack changer (Rack Changer II), two pumps (LC-30AD) and a column oven ( CTO-30A) composition. The mass spectrometer was calibrated using AB-SCIEX APCI positive and negative calibration solutions (Framingham, MA). Samples obtained from incubation with hepatocytes were analyzed in positive and negative scan mode. The basic analytical methods and instrumental conditions are summarized below. Modification of spectrometer settings depends on the necessity of the analyte.

LC-MS/MS conditions:

Data analysis of activity data

Mass spectral data were acquired using AB-Sciex Analyst TF (version 1.5.1; Framingham, MA). Chromatograms and spectra were acquired using AB-Sciex PeakView (version 2.2.0.1; Framingham, MA). Comparison of relative peak areas of extracted ion chromatograms was based on ±0.0002 Da of the expected exact mass-to-charge ratio (m/z) for each target analyte.

result

No metabolites were detected by incubation with hepatocytes. Under the analytical conditions presented, Compound 1a exhibited a retention time of approximately 7.8 minutes. In negative scan mode, compound 1a shows deprotonated molecular ion m/z 745 (C ₂₄ H ₂₅ F ₂ N ₁₀ O ₈ P ₂ S ₂ ⁻ ) and double deprotonated molecular ion m/z 372 (C _{24H24F2N10O8P2S22- )} ^. _{_ _ _ _ _} Major MS/MS product ions were observed at m/z 533 (C ₁₉ H ₁₉ FN ₁₀ O ₄ PS ⁻ ) and m/z 186 (C ₉ H ₈ N ₅ ⁻ ). In positive scan mode, compound 1a shows protonated molecular ions m/z 747 (C ₂₄ H ₂₇ F ₂ N ₁₀ O ₈ P ₂ S ₂ ⁺ ) and m/z 651 (C ₂₄ H ₂₆ F ₂ N ₁₀ O ₆ PS ⁺ ), major MS/MS product ions at m/z 252 (C ₁₀ H ₁₁ FN ₅ O ₂ ⁺ ) and m/z 188 (C ₉ H ₁₀ N ₅ ⁺ ). MS and MS/MS data confirmed the structure of compound 1a.

Compound 1a is stable in incubation with mouse, rat, dog, monkey and human hepatocytes. No obvious metabolite of compound 1a was found in this study. Among the samples obtained by incubation with hepatocytes, only compound 1a itself could be detected and confirmed by fragmentation by tandem mass spectrometry (MS/MS).

All documents cited in this disclosure are incorporated herein by reference, but to the extent that any incorporated document contradicts the written specification, the written specification shall control. Those skilled in the art will recognize that various changes and modifications of the material presented herein can be made and are within the scope and spirit of the present disclosure.

sequence listing

SEQ ID NO: 1 (WT human STING):

atgccccactccagcctgcatccatccatcccgtgtcccaggggtcacggggcccagaaggcagccttggttctgctgagtgcctgcctggtgaccctttgggggctaggagagccaccagagcacactctccggtacctggtgctccacctagcctccctgcagctgggactgctgttaaacggggtctgcagcctggctgaggagctgcgccacatccactccaggtaccggggcagctactggaggactgtgcgggcctgcctgggctgccccctccgccgtggggccctgttgctgctgtccatctatttctactactccctcccaaatgcggtcggcccgcccttcacttggatgcttgccctcctgggcctctcgcaggcactgaacatcctcctgggcctcaagggcctggccccagctgagatctctgcagtgtgtgaaaaagggaatttcaacgtggcccatgggctggcatggtcatattacatcggatatctgcggctgatcctgccagagctccaggcccggattcgaacttacaatcagcattacaacaacctgctacggggtgcagtgagccagcggctgtatattctcctcccattggactgtggggtgcctgataacctgagtatggctgaccccaacattcgcttcctggataaactgccccagcagaccggtgaccgggctggcatcaaggatcgggtttacagcaacagcatctatgagcttctggagaacgggcagcgggcgggcacctgtgtcctggagtacgccacccccttgcagactttgtttgccatgtcacaatacagtcaagctggctttagccgggaggataggcttgagcaggccaaactcttctgccggacacttgaggacatcctggcagatgcccctgagtctcagaacaactgccgcctcattgcctaccaggaacctgcagatgacagcagcttctcgctgtcccaggaggttctccggcacctgc ggcaggaggaaaaggaagaggttactgtgggcagcttgaagacctcagcggtgcccagtacctccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaagcccctccctctccgcacggatttctcttga

SEQ ID NO: 2 (REF human STING):

atgccccactccagcctgcatccatccatcccgtgtcccaggggtcacggggcccagaaggcagccttggttctgctgagtgcctgcctggtgaccctttgggggctaggagagccaccagagcacactctccggtacctggtgctccacctagcctccctgcagctgggactgctgttaaacggggtctgcagcctggctgaggagctgcgccacatccactccaggtaccggggcagctactggaggactgtgcgggcctgcctgggctgccccctccgccgtggggccctgttgctgctgtccatctatttctactactccctcccaaatgcggtcggcccgcccttcacttggatgcttgccctcctgggcctctcgcaggcactgaacatcctcctgggcctcaagggcctggccccagctgagatctctgcagtgtgtgaaaaagggaatttcaacgtggcccatgggctggcatggtcatattacatcggatatctgcggctgatcctgccagagctccaggcccggattcgaacttacaatcagcattacaacaacctgctacggggtgcagtgagccagcggctgtatattctcctcccattggactgtggggtgcctgataacctgagtatggctgaccccaacattcgcttcctggataaactgccccagcagaccggtgaccatgctggcatcaaggatcgggtttacagcaacagcatctatgagcttctggagaacgggcagcgggcgggcacctgtgtcctggagtacgccacccccttgcagactttgtttgccatgtcacaatacagtcaagctggctttagccgggaggataggcttgagcaggccaaactcttctgccggacacttgaggacatcctggcagatgcccctgagtctcagaacaactgccgcctcattgcctaccaggaacctgcagatgacagcagcttctcgctgtcccaggaggttctccggcacctgc ggcaggaggaaaaggaagaggttactgtgggcagcttgaagacctcagcggtgcccagtacctccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaagcccctccctctccgcacggatttctcttga

SEQ ID NO: 3 (AQ human STING):

atgccccactccagcctgcatccatccatcccgtgtcccaggggtcacggggcccagaaggcagccttggttctgctgagtgcctgcctggtgaccctttgggggctaggagagccaccagagcacactctccggtacctggtgctccacctagcctccctgcagctgggactgctgttaaacggggtctgcagcctggctgaggagctgcgccacatccactccaggtaccggggcagctactggaggactgtgcgggcctgcctgggctgccccctccgccgtggggccctgttgctgctgtccatctatttctactactccctcccaaatgcggtcggcccgcccttcacttggatgcttgccctcctgggcctctcgcaggcactgaacatcctcctgggcctcaagggcctggccccagctgagatctctgcagtgtgtgaaaaagggaatttcaacgtggcccatgggctggcatggtcatattacatcggatatctgcggctgatcctgccagagctccaggcccggattcgaacttacaatcagcattacaacaacctgctacggggtgcagtgagccagcggctgtatattctcctcccattggactgtggggtgcctgataacctgagtatggctgaccccaacattcgcttcctggataaactgccccagcagaccgctgaccgagctggcatcaaggatcgggtttacagcaacagcatctatgagcttctggagaacgggcagcgggcgggcacctgtgtcctggagtacgccacccccttgcagactttgtttgccatgtcacaatacagtcaagctggctttagccgggaggataggcttgagcaggccaaactcttctgccagacacttgaggacatcctggcagatgcccctgagtctcagaacaactgccgcctcattgcctaccaggaacctgcagatgacagcagcttctcgctgtcccaggaggttctccggcacctgc ggcaggaggaaaaggaagaggttactgtgggcagcttgaagacctcagcggtgcccagtacctccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaagcccctccctctccgcacggatttctcttga

SEQ ID NO: 4 (WT STING residues 155-341):

VAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDRAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEV

SEQ ID NO: 5 (His-TEV-Sumo-WT STING 155-341)

MHHHHHHSSGVDLGTENLYFQSNAMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGGSVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDRAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEV

SEQ ID NO: 6 (REF STING residues 155-341):

VAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEV

SEQ ID NO: 7 (His-TEV-Sumo-REF STING 155-341)

MHHHHHHSSGVDLGTENLYFQSNAMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGGSVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEV

sequence listing

<110> Eisai R&D Management Co., Ltd.

golden style

Fang Jiafan

Endo Atsushi

Choi Hyung Wook

Hao Minghong

Bao Xingfeng

Huang Guanqun

<120> Cyclic dinucleotide derivatives for the treatment of cancer

<130> 0080171-000378

<150> 62/460562

<151> 2017-02-17

<150> 62/479169

<151> 2017-03-30

<150> 62/551645

<151> 2017-08-29

<150> 62/551647

<151> 2017-08-29

<150> 62/551668

<151> 2017-08-29

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 1140

<212> DNA

<213> Homo sapiens

<400> 1

atgccccact ccagcctgca tccatccatc ccgtgtccca ggggtcacgg ggcccagaag 60

gcagccttgg ttctgctgag tgcctgcctg gtgacccttt gggggctagg agagccacca 120

gagcacactc tccggtacct ggtgctccac ctagcctccc tgcagctggg actgctgtta 180

aacggggtct gcagcctggc tgaggagctg cgccacatcc actccaggta ccggggcagc 240

tactggagga ctgtgcgggc ctgcctgggc tgccccctcc gccgtggggc cctgttgctg 300

ctgtccatct atttctacta ctccctccca aatgcggtcg gcccgccctt cacttggatg 360

cttgccctcc tgggcctctc gcaggcactg aacatcctcc tgggcctcaa gggcctggcc 420

ccagctgaga tctctgcagt gtgtgaaaaa gggaatttca acgtggccca tgggctggca 480

tggtcatatt acatcggata tctgcggctg atcctgccag agctccaggc ccggattcga 540

acttacaatc agcattacaa caacctgcta cggggtgcag tgagccagcg gctgtatatt 600

ctcctcccat tggactgtgg ggtgcctgat aacctgagta tggctgaccc caacattcgc 660

ttcctggata aactgcccca gcagaccggt gaccgggctg gcatcaagga tcgggtttac 720

agcaacagca tctatgagct tctggagaac gggcagcggg cgggcacctg tgtcctggag 780

tacgccaccc ccttgcagac tttgtttgcc atgtcacaat acagtcaagc tggctttagc 840

cgggaggata ggcttgagca ggccaaactc ttctgccgga cacttgagga catcctggca 900

gatgcccctg agtctcagaa caactgccgc ctcattgcct accaggaacc tgcagatgac 960

agcagcttct cgctgtccca ggaggttctc cggcacctgc ggcaggagga aaaggaagag 1020

gttactgtgg gcagcttgaa gacctcagcg gtgcccagta cctccacgat gtcccaagag 1080

cctgagctcc tcatcagtgg aatggaaaag cccctccctc tccgcacgga tttctcttga 1140

<210> 2

<211> 1140

<212> DNA

<213> Homo sapiens

<400> 2

atgccccact ccagcctgca tccatccatc ccgtgtccca ggggtcacgg ggcccagaag 60

gcagccttgg ttctgctgag tgcctgcctg gtgacccttt gggggctagg agagccacca 120

gagcacactc tccggtacct ggtgctccac ctagcctccc tgcagctggg actgctgtta 180

aacggggtct gcagcctggc tgaggagctg cgccacatcc actccaggta ccggggcagc 240

tactggagga ctgtgcgggc ctgcctgggc tgccccctcc gccgtggggc cctgttgctg 300

ctgtccatct atttctacta ctccctccca aatgcggtcg gcccgccctt cacttggatg 360

cttgccctcc tgggcctctc gcaggcactg aacatcctcc tgggcctcaa gggcctggcc 420

ccagctgaga tctctgcagt gtgtgaaaaa gggaatttca acgtggccca tgggctggca 480

tggtcatatt acatcggata tctgcggctg atcctgccag agctccaggc ccggattcga 540

acttacaatc agcattacaa caacctgcta cggggtgcag tgagccagcg gctgtatatt 600

ctcctcccat tggactgtgg ggtgcctgat aacctgagta tggctgaccc caacattcgc 660

ttcctggata aactgcccca gcagaccggt gaccatgctg gcatcaagga tcgggtttac 720

agcaacagca tctatgagct tctggagaac gggcagcggg cgggcacctg tgtcctggag 780

tacgccaccc ccttgcagac tttgtttgcc atgtcacaat acagtcaagc tggctttagc 840

cgggaggata ggcttgagca ggccaaactc ttctgccgga cacttgagga catcctggca 900

gatgcccctg agtctcagaa caactgccgc ctcattgcct accaggaacc tgcagatgac 960

agcagcttct cgctgtccca ggaggttctc cggcacctgc ggcaggagga aaaggaagag 1020

gttactgtgg gcagcttgaa gacctcagcg gtgcccagta cctccacgat gtcccaagag 1080

cctgagctcc tcatcagtgg aatggaaaag cccctccctc tccgcacgga tttctcttga 1140

<210> 3

<211> 1140

<212> DNA

<213> Homo sapiens

<400> 3

atgccccact ccagcctgca tccatccatc ccgtgtccca ggggtcacgg ggcccagaag 60

gcagccttgg ttctgctgag tgcctgcctg gtgacccttt gggggctagg agagccacca 120

gagcacactc tccggtacct ggtgctccac ctagcctccc tgcagctggg actgctgtta 180

aacggggtct gcagcctggc tgaggagctg cgccacatcc actccaggta ccggggcagc 240

tactggagga ctgtgcgggc ctgcctgggc tgccccctcc gccgtggggc cctgttgctg 300

ctgtccatct atttctacta ctccctccca aatgcggtcg gcccgccctt cacttggatg 360

cttgccctcc tgggcctctc gcaggcactg aacatcctcc tgggcctcaa gggcctggcc 420

ccagctgaga tctctgcagt gtgtgaaaaa gggaatttca acgtggccca tgggctggca 480

tggtcatatt acatcggata tctgcggctg atcctgccag agctccaggc ccggattcga 540

acttacaatc agcattacaa caacctgcta cggggtgcag tgagccagcg gctgtatatt 600

ctcctcccat tggactgtgg ggtgcctgat aacctgagta tggctgaccc caacattcgc 660

ttcctggata aactgcccca gcagaccgct gaccgagctg gcatcaagga tcgggtttac 720

agcaacagca tctatgagct tctggagaac gggcagcggg cgggcacctg tgtcctggag 780

tacgccaccc ccttgcagac tttgtttgcc atgtcacaat acagtcaagc tggctttagc 840

cgggaggata ggcttgagca ggccaaactc ttctgccaga cacttgagga catcctggca 900

gatgcccctg agtctcagaa caactgccgc ctcattgcct accaggaacc tgcagatgac 960

agcagcttct cgctgtccca ggaggttctc cggcacctgc ggcaggagga aaaggaagag 1020

gttactgtgg gcagcttgaa gacctcagcg gtgcccagta cctccacgat gtcccaagag 1080

cctgagctcc tcatcagtgg aatggaaaag cccctccctc tccgcacgga tttctcttga 1140

<210> 4

<211> 187

<212> PRT

<213> Homo sapiens

<400> 4

Val Ala His Gly Leu Ala Trp Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu

1 5 10 15

Ile Leu Pro Glu Leu Gln Ala Arg Ile Arg Thr Tyr Asn Gln His Tyr

20 25 30

Asn Asn Leu Leu Arg Gly Ala Val Ser Gln Arg Leu Tyr Ile Leu Leu

35 40 45

Pro Leu Asp Cys Gly Val Pro Asp Asn Leu Ser Met Ala Asp Pro Asn

50 55 60

Ile Arg Phe Leu Asp Lys Leu Pro Gln Gln Thr Gly Asp Arg Ala Gly

65 70 75 80

Ile Lys Asp Arg Val Tyr Ser Asn Ser Ile Tyr Glu Leu Leu Glu Asn

85 90 95

Gly Gln Arg Ala Gly Thr Cys Val Leu Glu Tyr Ala Thr Pro Leu Gln

100 105 110

Thr Leu Phe Ala Met Ser Gln Tyr Ser Gln Ala Gly Phe Ser Arg Glu

115 120 125

Asp Arg Leu Glu Gln Ala Lys Leu Phe Cys Arg Thr Leu Glu Asp Ile

130 135 140

Leu Ala Asp Ala Pro Glu Ser Gln Asn Asn Cys Arg Leu Ile Ala Tyr

145 150 155 160

Gln Glu Pro Ala Asp Asp Ser Ser Phe Ser Leu Ser Gln Glu Val Leu

165 170 175

Arg His Leu Arg Gln Glu Glu Lys Glu Glu Val

180 185

<210> 5

<211> 311

<212> PRT

<213> Artificial sequences

<220>

<223> His-TEV-Sumo-WT STING construct

<400> 5

Met His His His His His Ser Ser Gly Val Asp Leu Gly Thr Glu

1 5 10 15

Asn Leu Tyr Phe Gln Ser Asn Ala Met Ser Asp Ser Glu Val Asn Gln

20 25 30

Glu Ala Lys Pro Glu Val Lys Pro Glu Val Lys Pro Glu Thr His Ile

35 40 45

Asn Leu Lys Val Ser Asp Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys

50 55 60

Lys Thr Thr Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln

65 70 75 80

Gly Lys Glu Met Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile

85 90 95

Gln Ala Asp Gln Thr Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile

100 105 110

Ile Glu Ala His Arg Glu Gln Ile Gly Gly Gly Ser Val Ala His Gly

115 120 125

Leu Ala Trp Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Glu

130 135 140

Leu Gln Ala Arg Ile Arg Thr Tyr Asn Gln His Tyr Asn Asn Leu Leu

145 150 155 160

Arg Gly Ala Val Ser Gln Arg Leu Tyr Ile Leu Leu Pro Leu Asp Cys

165 170 175

Gly Val Pro Asp Asn Leu Ser Met Ala Asp Pro Asn Ile Arg Phe Leu

180 185 190

Asp Lys Leu Pro Gln Gln Thr Gly Asp Arg Ala Gly Ile Lys Asp Arg

195 200 205

Val Tyr Ser Asn Ser Ile Tyr Glu Leu Leu Glu Asn Gly Gln Arg Ala

210 215 220

Gly Thr Cys Val Leu Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala

225 230 235 240

Met Ser Gln Tyr Ser Gln Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu

245 250 255

Gln Ala Lys Leu Phe Cys Arg Thr Leu Glu Asp Ile Leu Ala Asp Ala

260 265 270

Pro Glu Ser Gln Asn Asn Cys Arg Leu Ile Ala Tyr Gln Glu Pro Ala

275 280 285

Asp Asp Ser Ser Phe Ser Leu Ser Gln Glu Val Leu Arg His Leu Arg

290 295 300

Gln Glu Glu Lys Glu Glu Val

305 310

<210> 6

<211> 187

<212> PRT

<213> Homo sapiens

<400> 6

Val Ala His Gly Leu Ala Trp Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu

1 5 10 15

Ile Leu Pro Glu Leu Gln Ala Arg Ile Arg Thr Tyr Asn Gln His Tyr

20 25 30

Asn Asn Leu Leu Arg Gly Ala Val Ser Gln Arg Leu Tyr Ile Leu Leu

35 40 45

Pro Leu Asp Cys Gly Val Pro Asp Asn Leu Ser Met Ala Asp Pro Asn

50 55 60

Ile Arg Phe Leu Asp Lys Leu Pro Gln Gln Thr Gly Asp His Ala Gly

65 70 75 80

Ile Lys Asp Arg Val Tyr Ser Asn Ser Ile Tyr Glu Leu Leu Glu Asn

85 90 95

Gly Gln Arg Ala Gly Thr Cys Val Leu Glu Tyr Ala Thr Pro Leu Gln

100 105 110

Thr Leu Phe Ala Met Ser Gln Tyr Ser Gln Ala Gly Phe Ser Arg Glu

115 120 125

Asp Arg Leu Glu Gln Ala Lys Leu Phe Cys Arg Thr Leu Glu Asp Ile

130 135 140

Leu Ala Asp Ala Pro Glu Ser Gln Asn Asn Cys Arg Leu Ile Ala Tyr

145 150 155 160

Gln Glu Pro Ala Asp Asp Ser Ser Phe Ser Leu Ser Gln Glu Val Leu

165 170 175

Arg His Leu Arg Gln Glu Glu Lys Glu Glu Val

180 185

<210> 7

<211> 311

<212> PRT

<213> Artificial sequences

<220>

<223> His-TEV-Sumo-REF STING 155-341 construct

<400> 7

Met His His His His His Ser Ser Gly Val Asp Leu Gly Thr Glu

1 5 10 15

Asn Leu Tyr Phe Gln Ser Asn Ala Met Ser Asp Ser Glu Val Asn Gln

20 25 30

Glu Ala Lys Pro Glu Val Lys Pro Glu Val Lys Pro Glu Thr His Ile

35 40 45

Asn Leu Lys Val Ser Asp Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys

50 55 60

Lys Thr Thr Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln

65 70 75 80

Gly Lys Glu Met Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile

85 90 95

Gln Ala Asp Gln Thr Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile

100 105 110

Ile Glu Ala His Arg Glu Gln Ile Gly Gly Gly Ser Val Ala His Gly

115 120 125

Leu Ala Trp Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Glu

130 135 140

Leu Gln Ala Arg Ile Arg Thr Tyr Asn Gln His Tyr Asn Asn Leu Leu

145 150 155 160

Arg Gly Ala Val Ser Gln Arg Leu Tyr Ile Leu Leu Pro Leu Asp Cys

165 170 175

Gly Val Pro Asp Asn Leu Ser Met Ala Asp Pro Asn Ile Arg Phe Leu

180 185 190

Asp Lys Leu Pro Gln Gln Thr Gly Asp His Ala Gly Ile Lys Asp Arg

195 200 205

Val Tyr Ser Asn Ser Ile Tyr Glu Leu Leu Glu Asn Gly Gln Arg Ala

210 215 220

Gly Thr Cys Val Leu Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala

225 230 235 240

Met Ser Gln Tyr Ser Gln Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu

245 250 255

Gln Ala Lys Leu Phe Cys Arg Thr Leu Glu Asp Ile Leu Ala Asp Ala

260 265 270

Pro Glu Ser Gln Asn Asn Cys Arg Leu Ile Ala Tyr Gln Glu Pro Ala

275 280 285

Asp Asp Ser Ser Phe Ser Leu Ser Gln Glu Val Leu Arg His Leu Arg

290 295 300

Gln Glu Glu Lys Glu Glu Val

305 310

Claims (1)

1. A compound having the formula: